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  • Tissue transglutaminase 2 regulates tumor cell tensional homeostasis by increasing contractility
    J. Cell Sci. (IF 4.401) Pub Date : 2020-01-03
    Bordeleau, F., Wang, W., Simmons, A., Antonyak, M. A., Cerione, R. A., Reinhart-King, C. A.

    Francois Bordeleau, Wenjun Wang, Alysha Simmons, Marc A. Antonyak, Richard A. Cerione, and Cynthia A. Reinhart-King Abnormal tensional cellular homeostasis is now considered a hallmark of cancer. Despite this, the origin of this abnormality remains unclear. In this work, we investigated the role of tissue transglutaminase 2 (TG2, also known as TGM2), a protein associated with poor prognosis and increased metastatic potential, and its relationship to the EGF receptor in the regulation of the mechanical state of tumor cells. Remarkably, we observed a TG2-mediated modulation of focal adhesion composition as well as stiffness-induced FAK activation, which was linked with a distinctive increase in cell contractility, in experiments using both pharmacological and shRNA-based approaches. Additionally, the increased contractility could be reproduced in non-malignant cells upon TG2 expression. Moreover, the increased cell contractility mediated by TG2 was largely due to the loss of EGFR-mediated inhibition of cell contractility. These findings establish intracellular TG2 as a regulator of cellular tensional homeostasis and suggest the existence of signaling switches that control the contribution of growth factor receptors in determining the mechanical state of a cell.

    更新日期:2020-01-14
  • Rhinovirus 2A is the key protease responsible for instigating the early block to gene expression in infected cells
    J. Cell Sci. (IF 4.401) Pub Date : 2020-01-07
    Smart, D., Filippi, I., Blume, C., Smalley, B., Davies, D., McCormick, C. J.

    David Smart, Irene Filippi, Cornelia Blume, Benjamin Smalley, Donna Davies, and Christopher J. McCormick Human rhinoviruses (HRVs) express 2 cysteine proteases, 2A and 3C, that are responsible for viral polyprotein processing. Both proteases also suppress host gene expression by inhibiting mRNA transcription, nuclear export and cap-dependent translation. However, the relative contribution that each makes in achieving this goal remains unclear. In this study, we have compared both the combined and individual ability of the two proteases to shut down cellular gene expression using a novel dynamic reporter system. Our findings show that 2A inhibits host gene expression much more rapidly than 3C. By comparing the activities of a representative set of proteases from the three different HRV species, we also find variation in the speed at which host gene expression is suppressed. Our work highlights the key role that 2A plays in early suppression of the infected host cell response and shows that this can be influenced by natural variation in the activity of this enzyme.

    更新日期:2020-01-14
  • Recruitment of integrin ανβ3 to integrin α5β1-induced clusters enables focal adhesion maturation and cell spreading.
    J. Cell Sci. (IF 4.401) Pub Date : null
    Carolina Diaz,Stefanie Neubauer,Florian Rechenmacher,Horst Kessler,Dimitris Missirlis

    The major fibronectin (FN) binding integrins α5β1 and αvβ3 exhibit cooperativity during cell adhesion, migration and mechanosensing, through mechanisms that are not yet fully resolved. Exploiting mechanically-tunable, nano-patterned substrates, and peptidomimetic ligands designed to selectively bind corresponding integrins, we report that focal adhesions (FAs) of endothelial cells assembled on integrin α5β1-selective substrates, rapidly recruit αvβ3 integrins, but not vice versa Blocking of integrin αvβ3 hindered FA maturation and cell spreading on α5β1-selective substrates, indicating a mechanism dependent on extracellular ligand binding and highlighting the requirement of αvβ3 engagement for efficient adhesion. Recruitment of αvβ3 integrins additionally occurred on hydrogel substrates of varying mechanical properties, above a threshold stiffness supporting FA formation. Mechanistic studies revealed the need for soluble factors present in serum to allow recruitment, and excluded exogenous, or endogenous, FN as the responsible ligand for integrin αvβ3 accumulation to adhesion clusters. Our findings highlight a novel mechanism of integrin co-operation and the critical role for αvβ3 integrins in promoting cell adhesion on α5β1-selective substrates.

    更新日期:2020-01-14
  • Plasmodium falciparum GCN5 acetyltransferase follows a novel proteolytic processing pathway that is essential for its function
    J. Cell Sci. (IF 4.401) Pub Date : 2020-01-09
    Bhowmick, K., Tehlan, A., Sunita, , Sudhakar, R., Kaur, I., Sijwali, P. S., Krishnamachari, A., Dhar, S. K.

    Krishanu Bhowmick, Ankita Tehlan, Sunita, Renu Sudhakar, Inderjeet Kaur, Puran Singh Sijwali, Annangarachari Krishnamachari, and Suman Kumar Dhar The pathogenesis of human malarial parasite Plasmodium falciparum is interlinked with its timely control of gene expression during its complex life cycle. In this organism, gene expression is partially controlled through epigenetic mechanisms, the regulation of which is, hence, of paramount importance to the parasite. The P. falciparum (Pf)-GCN5 histone acetyltransferase (HAT), an essential enzyme, acetylates histone 3 and regulates global gene expression in the parasite. Here, we show the existence of a novel proteolytic processing for PfGCN5 that is crucial for its activity in vivo. We find that a cysteine protease-like enzyme is required for the processing of PfGCN5 protein. Immunofluorescence and immuno-electron microscopy analysis suggest that the processing event occurs in the vicinity of the digestive vacuole of the parasite following its trafficking through the classical ER-Golgi secretory pathway, before it subsequently reaches the nucleus. Furthermore, blocking of PfGCN5 processing leads to the concomitant reduction of its occupancy at the gene promoters and a reduced H3K9 acetylation level at these promoters, highlighting the important correlation between the processing event and PfGCN5 activity. Altogether, our study reveals a unique processing event for a nuclear protein PfGCN5 with unforeseen role of a food vacuolar cysteine protease. This leads to a possibility of the development of new antimalarials against these targets. This article has an associated First Person interview with the first author of the paper.

    更新日期:2020-01-14
  • The inactivation domain of STIM1 acts through intramolecular binding to the coiled-coil domain in the resting state
    J. Cell Sci. (IF 4.401) Pub Date : 2020-01-08
    Lee, S. K., Lee, M.-h., Jeong, S. J., Qin, X., Lee, A. R., Park, H., Park, C. Y.

    Sang Kwon Lee, Min-hsun Lee, Su Ji Jeong, Xianan Qin, Ah Reum Lee, Hyokeun Park, and Chan Young Park Store-operated Ca2+ entry (SOCE) is a major Ca2+ influx pathway that is controlled by the ER Ca2+ sensor STIM1. Abnormal activation of STIM1 directly influences Ca2+ influx, resulting in severe diseases such as Stormorken syndrome. The inactivation domain of STIM1 (IDstim) has been identified as being essential for Ca2+-dependent inactivation of STIM1 (CDI) after SOCE occurs. However, it is unknown whether IDstim is involved in keeping STIM1 inactive before CDI. Herein, we show that IDstim helps STIM1 keep inactive through intramolecular binding with the coiled-coil domain. Between IDstim and the coiled-coil domain, we found a short conserved linker whose extension or mutation leads to the constitutive activation of STIM1. We have demonstrated that IDstim needs the coiled-coil domain 1 (CC1) to inhibit the Ca2+ release-activated Ca2+ (CRAC) activation domain (CAD) activity and binds to a CC1-CAD fragment. Serial deletion of CC1 revealed that CC1α1 is a co-inhibitory domain of IDstim. CC1α1 deletion or leucine mutation, which abolishes the closed conformation, impaired the inhibitory effect and binding of IDstim. These results suggest that IDstim cooperates with CC1α1 to help STIM1 keep inactive under resting conditions.

    更新日期:2020-01-14
  • Detecting single ORAI1 proteins within the plasma membrane reveals higher-order channel complexes
    J. Cell Sci. (IF 4.401) Pub Date : 2020-01-03
    Alansary, D., Peckys, D. B., Niemeyer, B. A., de Jonge, N.

    Dalia Alansary, Diana B. Peckys, Barbara A. Niemeyer, and Niels de Jonge ORAI1 proteins form highly selective Ca2+ channels in the plasma membrane. Crystallographic data point towards a hexameric stoichiometry of ORAI1 channels, whereas optical methods postulated ORAI1 channels to reside as dimers at rest, and other data suggests that they have a tetrameric configuration. Here, liquid-phase scanning transmission electron microscopy (STEM) and quantum dot (QD) labeling was utilized to study the conformation of ORAI1 proteins at rest. To address the question of whether ORAI1 was present as a dimer, experiments were designed using single ORAI1 monomers and covalently linked ORAI1 dimers with either one or two label-binding positions. The microscopic data was statistically analyzed via the pair correlation function. Label pairs were found in all cases, even for concatenated dimers with one label-binding position, which is only possible if a significant fraction of ORAI1 was assembled in larger order oligomers than dimers, binding at least two QDs. This interpretation of the data was consistent with Blue Native PAGE analysis showing that ORAI1 is mainly present as a complex of an apparent molecular mass larger than that calculated for a dimer.

    更新日期:2020-01-14
  • Welcoming early-career researchers to our Editorial Advisory Board
    J. Cell Sci. (IF 4.401) Pub Date : 2019-12-13
    Ahmad, S., Way, M.

    Sharon Ahmad and Michael Way

    更新日期:2019-12-30
  • The adaptor proteins HAP1a and GRIP1 collaborate to activate the kinesin-1 isoform KIF5C
    J. Cell Sci. (IF 4.401) Pub Date : 2019-12-13
    Twelvetrees, A. E., Lesept, F., Holzbaur, E. L. F., Kittler, J. T.

    Alison E. Twelvetrees, Flavie Lesept, Erika L. F. Holzbaur, and Josef T. Kittler Binding of motor proteins to cellular cargoes is regulated by adaptor proteins. HAP1 and GRIP1 are kinesin-1 adaptors that have been implicated individually in the transport of vesicular cargoes in the dendrites of neurons. We find that HAP1a and GRIP1 form a protein complex in the brain, and co-operate to activate the kinesin-1 subunit KIF5C in vitro. Based upon this co-operative activation of kinesin-1, we propose a modification to the kinesin activation model that incorporates stabilisation of the central hinge region known to be critical to autoinhibition of kinesin-1.

    更新日期:2019-12-30
  • The balance of forces generated by kinesins controls spindle polarity and chromosomal heterogeneity in tetraploid cells
    J. Cell Sci. (IF 4.401) Pub Date : 2019-12-13
    Shu, S., Iimori, M., Wakasa, T., Ando, K., Saeki, H., Oda, Y., Oki, E., Maehara, Y.

    Sei Shu, Makoto Iimori, Takeshi Wakasa, Koji Ando, Hiroshi Saeki, Yoshinao Oda, Eiji Oki, and Yoshihiko Maehara Chromosomal instability, one of the most prominent features of tumour cells, causes aneuploidy. Tetraploidy is thought to be an intermediate on the path to aneuploidy, but the mechanistic relationship between the two states is poorly understood. Here, we show that spindle polarity (e.g. bipolarity or multipolarity) in tetraploid cells depends on the level of functional phosphorylated Eg5, a mitotic kinesin, localised to the spindle. Multipolar spindles are formed in cells with high levels of phosphorylated Eg5. This process is suppressed by inhibition of Eg5 or expression of a non-phosphorylatable Eg5 mutant, as well as by changing the balance between opposing forces required for centrosome separation. Tetraploid cells with high levels of functional Eg5 give rise to a heterogeneous aneuploid population through multipolar division, whereas cells with low levels of functional Eg5 continue to undergo bipolar division and remain tetraploid. Furthermore, Eg5 protein levels correlate with ploidy status in tumour specimens. We provide a novel explanation for the tetraploid intermediate model, i.e. spindle polarity and subsequent tetraploid cell behaviour are determined by the balance of forces generated by mitotic kinesins at the spindle.

    更新日期:2019-12-30
  • Concerted regulation of actin polymerization during constitutive secretion by cortactin and PKD2
    J. Cell Sci. (IF 4.401) Pub Date : 2019-12-13
    Weeber, F., Becher, A., Seibold, T., Seufferlein, T., Eiseler, T.

    Florian Weeber, Alexander Becher, Tanja Seibold, Thomas Seufferlein, and Tim Eiseler Constitutive secretion from the trans-Golgi-network (TGN) is facilitated by a concerted regulation of vesicle biogenesis and fission processes. The protein kinase D family (PKD) has been previously described to enhance vesicle fission by modifying the lipid environment. PKD also phosphorylates the actin regulatory protein cortactin at S298 to impair synergistic actin polymerization. We here report additional functions for PKD2 (also known as PRKD2) and cortactin in the regulation of actin polymerization during the fission of transport carriers from the TGN. Phosphorylation of cortactin at S298 impairs the interaction between WIP (also known as WIPF1) and cortactin. WIP stabilizes the autoinhibited conformation of N-WASP (also known as WASL). This leads to an inhibition of synergistic Arp2/3-complex-dependent actin polymerization at the TGN. PKD2 activity at the TGN is controlled by active CDC42-GTP which directly activates N-WASP, inhibits PKD2 and shifts the balance to non-S298-phosphorylated cortactin, which can in turn sequester WIP from N-WASP. Consequently, synergistic actin polymerization at the TGN and constitutive secretion are enhanced.

    更新日期:2019-12-30
  • A retention-release mechanism based on RAB11FIP2 for AMPA receptor synaptic delivery during long-term potentiation
    J. Cell Sci. (IF 4.401) Pub Date : 2019-12-16
    Royo, M., Gutierrez, Y., Fernandez-Monreal, M., Gutierrez-Eisman, S., Jimenez, R., Jurado, S., Esteban, J. A.

    Maria Royo, Yolanda Gutierrez, Monica Fernandez-Monreal, Silvia Gutierrez-Eisman, Raquel Jimenez, Sandra Jurado, and Jose A. Esteban It is well­-established that Rab11-dependent recycling endosomes drive the activity-dependent delivery of AMPA receptors (AMPARs) into synapses during long-term potentiation (LTP). Nevertheless, the molecular basis for this specialized function of recycling endosomes is still unknown. Here, we have investigated RAB11FIP2 (FIP2 hereafter) as a potential effector of Rab11-dependent trafficking during LTP in rat hippocampal slices. Surprisingly, we found that FIP2 operates independently from Rab11 proteins, and acts as a negative regulator of AMPAR synaptic trafficking. Under basal conditions, FIP2 associates with AMPARs at immobile compartments, separately from recycling endosomes. Using shRNA-mediated knockdown, we found that FIP2 prevents GluA1 (encoded by the Gria1 gene) AMPARs from reaching the surface of dendritic spines in the absence of neuronal stimulation. Upon induction of LTP, FIP2 is rapidly mobilized, dissociates from AMPARs and undergoes dephosphorylation. Interestingly, this dissociation of the FIP2–AMPAR complex, together with FIP2 dephosphorylation, is required for LTP, but the interaction between FIP2 and Rab11 proteins is not. Based on these results, we propose a retention–release mechanism, where FIP2 acts as a gate that restricts the trafficking of AMPARs, until LTP induction triggers their release and allows synaptic delivery.

    更新日期:2019-12-30
  • Melanoma mutations modify melanocyte dynamics in co-culture with keratinocytes or fibroblasts
    J. Cell Sci. (IF 4.401) Pub Date : 2019-12-13
    Skalamera, D., Stevenson, A. J., Ehmann, A., Ainger, S. A., Lanagan, C., Sturm, R. A., Gabrielli, B.

    Dubravka Skalamera, Alexander J. Stevenson, Anna Ehmann, Stephen A. Ainger, Catherine Lanagan, Richard A. Sturm, and Brian Gabrielli Melanocytic cell interactions are integral to skin homeostasis, and affect the outcome of multiple diseases, including cutaneous pigmentation disorders and melanoma. By using automated-microscopy and machine-learning-assisted morphology analysis of primary human melanocytes in co-culture, we performed combinatorial interrogation of melanocyte genotypic variants and functional assessment of lentivirus-introduced mutations. Keratinocyte-induced melanocyte dendricity, an indicator of melanocyte differentiation, was reduced in the melanocortin 1 receptor (MC1R) R/R variant strain and by NRAS.Q61K and BRAF.V600E expression, while expression of CDK4.R24C and RAC1.P29S had no detectable effect. Time-lapse tracking of melanocytes in co-culture revealed dynamic interaction phenotypes and hyper-motile cell states that indicated that, in addition to the known role in activating mitogenic signalling, MEK-pathway-activating mutations may also allow melanocytes to escape keratinocyte control and increase their invasive potential. Expanding this combinatorial platform will identify other therapeutic target mutations and melanocyte genetic variants, as well as increase understanding of skin cell interactions.

    更新日期:2019-12-30
  • The spindle pole body of Aspergillus nidulans is asymmetrical and contains changing numbers of {gamma}-tubulin complexes
    J. Cell Sci. (IF 4.401) Pub Date : 2019-12-13
    Gao, X., Schmid, M., Zhang, Y., Fukuda, S., Takeshita, N., Fischer, R.

    Xiaolei Gao, Marjorie Schmid, Ying Zhang, Sayumi Fukuda, Norio Takeshita, and Reinhard Fischer Centrosomes are important microtubule-organizing centers (MTOCs) in animal cells. In addition, non-centrosomal MTOCs (ncMTOCs) are found in many cell types. Their composition and structure are only poorly understood. Here, we analyzed nuclear MTOCs (spindle-pole bodies, SPBs) and septal MTOCs in Aspergillus nidulans. They both contain -tubulin along with members of the family of -tubulin complex proteins (GCPs). Our data suggest that SPBs consist of -tubulin small complexes (-TuSCs) at the outer plaque, and larger -tubulin ring complexes (-TuRC) at the inner plaque. We show that the MztA protein, an ortholog of the human MOZART protein (also known as MZT1), interacted with the inner plaque receptor PcpA (the homolog of fission yeast Pcp1) at SPBs, while no interaction nor colocalization was detected between MztA and the outer plaque receptor ApsB (fission yeast Mto1). Septal MTOCs consist of -TuRCs including MztA but are anchored through AspB and Spa18 (fission yeast Mto2). MztA is not essential for viability, although abnormal spindles were observed frequently in cells lacking MztA. Quantitative PALM imaging revealed unexpected dynamics of the protein composition of SPBs, with changing numbers of -tubulin complexes over time during interphase and constant numbers during mitosis. This article has an associated First Person interview with the first author of the paper.

    更新日期:2019-12-30
  • Mark1 regulates distal airspace expansion through type I pneumocyte flattening in lung development
    J. Cell Sci. (IF 4.401) Pub Date : 2019-12-13
    Fumoto, K., Takigawa-Imamura, H., Sumiyama, K., Yoshimura, S. H., Maehara, N., Kikuchi, A.

    Katsumi Fumoto, Hisako Takigawa-Imamura, Kenta Sumiyama, Shige H. Yoshimura, Natsumi Maehara, and Akira Kikuchi During the later stages of lung development, two types of pneumocytes, cuboidal type II (AECII) and flattened type I (AECI) alveolar epithelial cells, form distal lung saccules. Here, we highlight how fibroblasts expressing MAP-microtubule affinity regulating kinase 1 (Mark1) are required for the terminal stages of pulmonary development, called lung sacculation. In Mark1-knockout (KO) mice, distal sacculation and AECI flattening are significantly impaired. Fetal epithelial cells generate alveolar organoids and differentiate into pneumocytes when co-cultured with fibroblasts. However, the size of organoids decreased and AECI flattening was impaired in the presence of Mark1 KO fibroblasts. In Mark1 KO fibroblasts themselves, cilia formation and the Hedgehog pathway were suppressed, resulting in the loss of type I collagen expression. The addition of type I collagen restored AECI flattening in organoids co-cultured with Mark1 KO fibroblasts and rescued the decreased size of organoids. Mathematical modeling of distal lung sacculation supports the view that AECI flattening is necessary for the proper formation of saccule-like structures. These results suggest that Mark1-mediated fibroblast activation induces AECI flattening and thereby regulates distal lung sacculation.

    更新日期:2019-12-30
  • An in vitro compartmental system underlines the contribution of mitochondrial immobility to the ATP supply in the NMJ
    J. Cell Sci. (IF 4.401) Pub Date : 2019-12-02
    Altman, T., Geller, D., Kleeblatt, E., Gradus-Perry, T., Perlson, E.

    Topaz Altman, Danielle Geller, Elisabeth Kleeblatt, Tal Gradus-Perry, and Eran Perlson The neuromuscular junction (NMJ) is the largest, most-complex synapse in the human body. Motor neuron (MN) diseases, such as amyotrophic lateral sclerosis (ALS), specifically target MNs and the NMJs. However, little is known about the reasons for MN-selective neuronal and synaptic vulnerability in MN diseases. Here, utilizing a compartmental microfluidic in vitro co-culture system, we provide a possible explanation for why the NMJ, other than its unusual dimensions, differs from other synapses. By using live-imaging techniques, we discovered that cultured MNs display higher axonal and synaptic mitochondrial immobility compared with sympathetic neurons (SNs), leading to a profound enrichment of mitochondria only in the MN NMJ. Furthermore, by employing a synaptic ATP sensor, we show that mitochondrial respiration is the key contributor to ATP production in MN NMJs but not in SN synapses. Taken together, our data suggest that mitochondrial localization underlies the unique and specific qualities of MN NMJs. Our findings shed light on the role of mitochondria in MN and NMJ maintenance, and possibly indicate how mitochondria may serve as a source for selective MN vulnerability in neurodegenerative diseases. This article has an associated First Person interview with the first author of the paper.

    更新日期:2019-12-13
  • High-density lipoprotein or cyclodextrin extraction of cholesterol from aggregated LDL reduces foam cell formation
    J. Cell Sci. (IF 4.401) Pub Date : 2019-12-02
    Singh, R. K., Lund, F. W., Haka, A. S., Maxfield, F. R.

    Rajesh K. Singh, Frederik W. Lund, Abigail S. Haka, and Frederick R. Maxfield Low-density lipoprotein (LDL) deposition, aggregation and retention in the endothelial sub-intima are critical initiating events during atherosclerosis. Macrophages digest aggregated LDL (agLDL) through a process called exophagy. High-density lipoprotein (HDL) plays an atheroprotective role, but studies attempting to exploit it therapeutically have been unsuccessful, highlighting gaps in our current understanding of HDL function. Here, we characterized the role of HDL during exophagy of agLDL. We find that atherosclerotic plaque macrophages contact agLDL and form an extracellular digestive compartment similar to that observed in vitro. During macrophage catabolism of agLDL in vitro, levels of free cholesterol in the agLDL are increased. HDL can extract free cholesterol directly from this agLDL and inhibit macrophage foam cell formation. Cholesterol-balanced hydroxypropyl-β-cyclodextrin similarly reduced macrophage cholesterol uptake and foam cell formation. Finally, we show that HDL can directly extract free cholesterol, but not cholesterol esters, from agLDL in the absence of cells. Together, these results suggest that the actions of HDL can directly extract free cholesterol from agLDL during catabolism, and provide a new context in which to view the complex relationship between HDL and atherosclerosis.

    更新日期:2019-12-13
  • Hmgcr promotes a long-range signal to attract Drosophila germ cells independently of Hedgehog
    J. Cell Sci. (IF 4.401) Pub Date : 2019-12-02
    Kenwrick, K., Mukherjee, A., Renault, A. D.

    Kim Kenwrick, Amrita Mukherjee, and Andrew D. Renault During development, many cell types migrate along stereotyped routes determined through deployment of cell surface or secreted guidance molecules. Although we know the identity of many of these molecules, the distances over which they natively operate can be difficult to determine. Here, we have quantified the range of an attractive signal for the migration of Drosophila germ cells. Their migration is guided by an attractive signal generated by the expression of genes in the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (Hmgcr) pathway, and by a repulsive signal generated by the expression of Wunens. We demonstrate that the attractive signal downstream of Hmgcr is cell-contact independent and acts at long range, the extent of which depends on Hmgcr levels. This range would be sufficient to reach all of the germ cells for their entire migration. Furthermore, Hmgcr-mediated attraction does not require Wunens but can operate simultaneously with Wunen-mediated repulsion. Finally, several papers posit Hedgehog (Hh) as being the germ cell attractant downstream of Hmgcr. Here, we provide evidence that this is not the case.

    更新日期:2019-12-13
  • MUNC18-1 regulates the submembrane F-actin network, independently of syntaxin1 targeting, via hydrophobicity in {beta}-sheet 10
    J. Cell Sci. (IF 4.401) Pub Date : 2019-12-02
    Pons-Vizcarra, M., Kurps, J., Tawfik, B., Sorensen, J. B., van Weering, J. R. T., Verhage, M.

    Maria Pons-Vizcarra, Julia Kurps, Bassam Tawfik, Jakob B. Sorensen, Jan R. T. van Weering, and Matthijs Verhage MUNC18-1 (also known as STXBP1) is an essential protein for docking and fusion of secretory vesicles. Mouse chromaffin cells (MCCs) lacking MUNC18-1 show impaired secretory vesicle docking, but also mistargeting of SNARE protein syntaxin1 and an abnormally dense submembrane F-actin network. Here, we tested the contribution of both these phenomena to docking and secretion defects in MUNC18-1-deficient MCCs. We show that an abnormal F-actin network and syntaxin1 targeting defects are not observed in Snap25- or Syt1-knockout (KO) MCCs, which are also secretion deficient. We identified a MUNC18-1 mutant (V263T in β-sheet 10) that fully restores syntaxin1 targeting but not F-actin abnormalities in Munc18-1-KO cells. MUNC18-2 and -3 (also known as STXBP2 and STXBP3, respectively), which lack the hydrophobic residue at position 263, also did not restore a normal F-actin network in Munc18-1-KO cells. However, these proteins did restore the normal F-actin network when a hydrophobic residue was introduced at the corresponding position. Munc18-1-KO MCCs expressing MUNC18-1(V263T) showed normal vesicle docking and exocytosis. These results demonstrate that MUNC18-1 regulates the F-actin network independently of syntaxin1 targeting via hydrophobicity in β-sheet 10. The abnormally dense F-actin network in Munc18-1-deficient cells is not a rate-limiting barrier in secretory vesicle docking or fusion. This article has an associated First Person interview with the first author of the paper.

    更新日期:2019-12-13
  • A novel interplay between GEFs orchestrates Cdc42 activity during cell polarity and cytokinesis in fission yeast
    J. Cell Sci. (IF 4.401) Pub Date : 2019-12-03
    Hercyk, B. S., Rich-Robinson, J., Mitoubsi, A. S., Harrell, M. A., Das, M. E.

    Brian S. Hercyk, Julie Rich-Robinson, Ahmad S. Mitoubsi, Marcus A. Harrell, and Maitreyi E. Das Cdc42, a conserved regulator of cell polarity, is activated by two GEFs, Gef1 and Scd1, in fission yeast. Why the cell needs two GEFs is unclear, given that they are partially redundant and activate the same GTPase. Using the GEF localization pattern during cytokinesis as a paradigm, we report a novel interplay between Gef1 and Scd1 that spatially modulates Cdc42. We find that Gef1 promotes Scd1 localization to the division site during cytokinesis through recruitment of the scaffold protein Scd2, via a Cdc42 feedforward pathway. Similarly, during interphase Gef1 promotes Scd1 recruitment at the new end to enable the transition from monopolar to bipolar growth. Reciprocally, Scd1 restricts Gef1 localization to prevent ectopic Cdc42 activation during cytokinesis to promote cell separation, and to maintain cell shape during interphase. Our findings reveal an elegant regulatory pattern in which Gef1 primes Cdc42 activation at new sites to initiate Scd1-dependent polarized growth, while Scd1 restricts Gef1 to sites of polarization. We propose that crosstalk between GEFs is a conserved mechanism that orchestrates Cdc42 activation during complex cellular processes. This article has an associated First Person interview with the first author of the paper.

    更新日期:2019-12-13
  • TRIM32, but not its muscular dystrophy-associated mutant, positively regulates and is targeted to autophagic degradation by p62/SQSTM1
    J. Cell Sci. (IF 4.401) Pub Date : 2019-12-02
    Overa, K. S., Garcia-Garcia, J., Bhujabal, Z., Jain, A., Overvatn, A., Larsen, K. B., Deretic, V., Johansen, T., Lamark, T., Sjottem, E.

    Katrine Stange Overa, Juncal Garcia-Garcia, Zambarlal Bhujabal, Ashish Jain, Aud Overvatn, Kenneth Bowitz Larsen, Vojo Deretic, Terje Johansen, Trond Lamark, and Eva Sjottem The tripartite motif (TRIM) proteins constitute a family of ubiquitin E3 ligases involved in a multitude of cellular processes, including protein homeostasis and autophagy. TRIM32 is characterized by six protein–protein interaction domains termed NHL, various point mutations in which are associated with limb-girdle-muscular dystrophy 2H (LGMD2H). Here, we show that TRIM32 is an autophagy substrate. Lysosomal degradation of TRIM32 was dependent on ATG7 and blocked by knockout of the five autophagy receptors p62 (also known as SQSTM1), NBR1, NDP52 (also known as CALCOCO2), TAX1BP1 and OPTN, pointing towards degradation by selective autophagy. p62 directed TRIM32 to lysosomal degradation, while TRIM32 mono-ubiquitylated p62 on lysine residues involved in regulation of p62 activity. Loss of TRIM32 impaired p62 sequestration, while reintroduction of TRIM32 facilitated p62 dot formation and its autophagic degradation. A TRIM32LGMD2H disease mutant was unable to undergo autophagic degradation and to mono-ubiquitylate p62, and its reintroduction into the TRIM32-knockout cells did not affect p62 dot formation. In light of the important roles of autophagy and p62 in muscle cell proteostasis, our results point towards impaired TRIM32-mediated regulation of p62 activity as a pathological mechanisms in LGMD2H.

    更新日期:2019-12-13
  • Mammalian TRP ion channels are insensitive to membrane stretch
    J. Cell Sci. (IF 4.401) Pub Date : 2019-12-10
    Nikolaev, Y. A., Cox, C. D., Ridone, P., Rohde, P. R., Cordero-Morales, J. F., Vasquez, V., Laver, D. R., Martinac, B.

    Yury A. Nikolaev, Charles D. Cox, Pietro Ridone, Paul R. Rohde, Julio F. Cordero-Morales, Valeria Vasquez, Derek R. Laver, and Boris Martinac TRP channels of the transient receptor potential ion channel superfamily are involved in a wide variety of mechanosensory processes, including touch sensation, pain, blood pressure regulation, bone loading and detection of cerebrospinal fluid flow. However, in many instances it is unclear whether TRP channels are the primary transducers of mechanical force in these processes. In this study, we tested stretch activation of eleven TRP channels from six mammalian subfamilies. We found that these TRP channels were insensitive to short membrane stretches in cellular systems. Furthermore, we purified TRPC6 and demonstrated its insensitivity to stretch in liposomes, an artificial bilayer system free from cellular components. Additionally, we demonstrated that, when expressed in C. elegans neurons, mouse TRPC6 restores the mechanoresponse of a touch insensitive mutant but requires diacylglycerol for activation. These results strongly suggest that the mammalian members of the TRP ion channel family are insensitive to tension induced by cell membrane stretching and, thus, are more likely to be activated by cytoplasmic tethers or downstream components and to act as amplifiers of cellular mechanosensory signaling cascades.

    更新日期:2019-12-13
  • The liquid nucleome - phase transitions in the nucleus at a glance
    J. Cell Sci. (IF 4.401) Pub Date : 2019-11-21
    Strom, A. R., Brangwynne, C. P.

    Amy R. Strom and Clifford P. Brangwynne Cells organize membrane-less internal compartments through a process called liquid–liquid phase separation (LLPS) to create chemically distinct compartments, referred to as condensates, which emerge from interactions among biological macromolecules. These condensates include various cytoplasmic structures such as P-granules and stress granules. However, an even wider array of condensates subcompartmentalize the cell nucleus, forming liquid-like structures that range from nucleoli and Cajal bodies to nuclear speckles and gems. Phase separation provides a biophysical assembly mechanism underlying this non-covalent form of fluid compartmentalization and functionalization. In this Cell Science at a Glance article and the accompanying poster, we term these phase-separated liquids that organize the nucleus the liquid nucleome; we discuss examples of biological phase transitions in the nucleus, how the cell utilizes biophysical aspects of phase separation to form and regulate condensates, and suggest interpretations for the role of phase separation in nuclear organization and function.

    更新日期:2019-11-29
  • Yap induces osteoblast differentiation by modulating Bmp signalling during zebrafish caudal fin regeneration
    J. Cell Sci. (IF 4.401) Pub Date : 2019-11-14
    Brandao, A. S., Bensimon-Brito, A., Lourenco, R., Borbinha, J., Soares, A. R., Mateus, R., Jacinto, A.

    Ana S. Brandao, Anabela Bensimon-Brito, Raquel Lourenco, Jorge Borbinha, Ana Rosa Soares, Rita Mateus, and Antonio Jacinto Osteoblast differentiation is a key process for bone homeostasis and repair. Multiple signalling pathways have been associated with osteoblast differentiation, yet much remains unknown on how this process is regulated in vivo. Previous studies have proposed that the Hippo pathway transcriptional co-activators YAP and TAZ (also known as YAP1 and WWTR1, respectively) maintain progenitor stemness and inhibit terminal differentiation of osteoblasts, whereas others suggest they potentiate osteoblast differentiation and bone formation. Here, we use zebrafish caudal fin regeneration as a model to clarify how the Hippo pathway regulates de novo bone formation and osteoblast differentiation. We demonstrate that Yap inhibition leads to accumulation of osteoprogenitors and prevents osteoblast differentiation in a cell non-autonomous manner. This effect correlates with a severe impairment of Bmp signalling in osteoblasts, likely by suppressing the expression of the ligand bmp2a in the surrounding mesenchymal cells. Overall, our findings provide a new mechanism of bone formation through the Hippo–Yap pathway, integrating Yap in the signalling cascade that governs osteoprogenitor maintenance and subsequent differentiation during zebrafish caudal fin regeneration.

    更新日期:2019-11-29
  • MICAL-L1 coordinates ciliogenesis by recruiting EHD1 to the primary cilium
    J. Cell Sci. (IF 4.401) Pub Date : 2019-11-14
    Xie, S., Farmer, T., Naslavsky, N., Caplan, S.

    Shuwei Xie, Trey Farmer, Naava Naslavsky, and Steve Caplan The endocytic protein EHD1 plays an important role in ciliogenesis by facilitating fusion of the ciliary vesicle and removal of CP110 (also known as CCP110) from the mother centriole, as well as removal of Cep215 (also known as CDK5RAP2) from centrioles to permit disengagement and duplication. However, the mechanism of its centrosomal recruitment remains unknown. Here, we address the role of the EHD1 interaction partner MICAL-L1 in ciliogenesis. MICAL-L1 knockdown impairs ciliogenesis in a similar manner to EHD1 knockdown, and MICAL-L1 localizes to cilia and centrosomes in both ciliated and non-ciliated cells. Consistent with EHD1 function, MICAL-L1-depletion prevents CP110 removal from the mother centriole. Moreover, upon MICAL-L1-depletion, EHD1 fails to localize to basal bodies. Since MICAL-L1 localizes to the centrosome even in non-ciliated cells, we hypothesized that it might be anchored to the centrosome via an interaction with centrosomal proteins. By performing mass spectrometry, we identified several tubulins as potential MICAL-L1 interaction partners, and found a direct interaction between MICAL-L1 and both α-tubulin–β-tubulin heterodimers and -tubulin. Our data support the notion that a pool of centriolar -tubulin and/or α-tubulin–β-tubulin heterodimers anchor MICAL-L1 to the centriole, where it might recruit EHD1 to promote ciliogenesis.

    更新日期:2019-11-29
  • A homeostatic mechanism rapidly corrects aberrant nucleocytoplasmic ratios maintaining nuclear size in fission yeast
    J. Cell Sci. (IF 4.401) Pub Date : 2019-11-14
    Cantwell, H., Nurse, P.

    Helena Cantwell and Paul Nurse Nuclear size scales with cell size across a wide range of cell types. The mechanism by which this scaling is maintained in growing cells remains unclear. Here, we investigate the mechanism of nuclear size homeostasis in the simple eukaryote fission yeast, by monitoring the recovery of aberrant nuclear volume to cell volume (N/C) ratios following perturbation. We demonstrate that both high and low N/C ratios correct rapidly, maintaining nuclear size homeostasis. We assess the kinetics of nuclear and cellular growth and of N/C ratio correction, and demonstrate that nuclear and cellular growth rates are not directly coupled. We propose that the mechanism underlying nuclear size homeostasis involves multiple limiting factors implicated in processes including nucleocytoplasmic transport, lipid biogenesis and RNA processing. We speculate that these link cellular size increases to changes in nuclear contents, which in turn lead to changes in nuclear membrane surface area. Our study reveals that there is rapid nuclear size homeostasis in cells, informing understanding of nuclear size control and size homeostasis of other membrane-bound organelles.

    更新日期:2019-11-29
  • Role for the splicing factor TCERG1 in Cajal body integrity and snRNP assembly
    J. Cell Sci. (IF 4.401) Pub Date : 2019-11-14
    Moreno-Castro, C., Prieto-Sanchez, S., Sanchez-Hernandez, N., Hernandez-Munain, C., Sune, C.

    Cristina Moreno-Castro, Silvia Prieto-Sanchez, Noemi Sanchez-Hernandez, Cristina Hernandez-Munain, and Carlos Sune Cajal bodies are nuclear organelles involved in the nuclear phase of small nuclear ribonucleoprotein (snRNP) biogenesis. In this study, we identified the splicing factor TCERG1 as a coilin-associated factor that is essential for Cajal body integrity. Knockdown of TCERG1 disrupts the localization of the components of Cajal bodies, including coilin and NOLC1, with coilin being dispersed in the nucleoplasm into numerous small foci, without affecting speckles, gems or the histone locus body. Furthermore, the depletion of TCERG1 affects the recruitment of Sm proteins to uridine-rich small nuclear RNAs (snRNAs) to form the mature core snRNP. Taken together, the results of this study suggest that TCERG1 plays an important role in Cajal body formation and snRNP biogenesis.

    更新日期:2019-11-29
  • Condensin II protein dysfunction impacts mitochondrial respiration and mitochondrial oxidative stress responses
    J. Cell Sci. (IF 4.401) Pub Date : 2019-11-20
    Deutschman, E., Ward, J. R., Kumar, A., Ray, G., Welch, N., Lemieux, M. E., Dasarathy, S., Longworth, M. S.

    Emily Deutschman, Jacqueline R. Ward, Avinash Kumar, Greeshma Ray, Nicole Welch, Madeleine E. Lemieux, Srinivisan Dasarathy, and Michelle S. Longworth The maintenance of mitochondrial respiratory function and homeostasis is essential to human health. Here, we identify condensin II subunits as novel regulators of mitochondrial respiration and mitochondrial stress responses. Condensin II is present in the nucleus and cytoplasm. While the effects of condensin II depletion on nuclear genome organization are well studied, the effects on essential cytoplasmic and metabolic processes are not as well understood. Excitingly, we observe that condensin II chromosome-associated protein (CAP) subunits individually localize to different regions of mitochondria, suggesting possible mitochondrial-specific functions independent from those mediated by the canonical condensin II holocomplex. Changes in cellular ATP levels and mitochondrial respiration are observed in condensin II CAP subunit-deficient cells. Surprisingly, we find that loss of NCAPD3 also sensitizes cells to oxidative stress. Together, these studies identify new, and possibly independent, roles for condensin II CAP subunits in preventing mitochondrial damage and dysfunction. These findings reveal a new area of condensin protein research that could contribute to the identification of targets to treat diseases where aberrant function of condensin II proteins is implicated.

    更新日期:2019-11-29
  • Vac8 spatially confines autophagosome formation at the vacuole in S. cerevisiae
    J. Cell Sci. (IF 4.401) Pub Date : 2019-11-14
    Hollenstein, D. M., Gomez-Sanchez, R., Ciftci, A., Kriegenburg, F., Mari, M., Torggler, R., Licheva, M., Reggiori, F., Kraft, C.

    David M. Hollenstein, Ruben Gomez-Sanchez, Akif Ciftci, Franziska Kriegenburg, Muriel Mari, Raffaela Torggler, Mariya Licheva, Fulvio Reggiori, and Claudine Kraft Autophagy is initiated by the formation of a phagophore assembly site (PAS), the precursor of autophagosomes. In mammals, autophagosome formation sites form throughout the cytosol in specialized subdomains of the endoplasmic reticulum (ER). In yeast, the PAS is also generated close to the ER, but always in the vicinity of the vacuole. How the PAS is anchored to the vacuole and the functional significance of this localization are unknown. Here, we investigated the role of the PAS–vacuole connection for bulk autophagy in the yeast Saccharomyces cerevisiae. We show that Vac8 constitutes a vacuolar tether that stably anchors the PAS to the vacuole throughout autophagosome biogenesis via the PAS component Atg13. S. cerevisiae lacking Vac8 show inefficient autophagosome–vacuole fusion, and form fewer and smaller autophagosomes that often localize away from the vacuole. Thus, the stable PAS–vacuole connection established by Vac8 creates a confined space for autophagosome biogenesis between the ER and the vacuole, and allows spatial coordination of autophagosome formation and autophagosome–vacuole fusion. These findings reveal that the spatial regulation of autophagosome formation at the vacuole is required for efficient bulk autophagy.

    更新日期:2019-11-29
  • Two conserved glycine residues in mammalian and Dictyostelium Rictor are required for mTORC2 activity and integrity
    J. Cell Sci. (IF 4.401) Pub Date : 2019-11-14
    Pergolizzi, B., Panuzzo, C., Ali, M. S., Lo Iacono, M., Levra Levron, C., Ponzone, L., Prelli, M., Cilloni, D., Calautti, E., Bozzaro, S., Bracco, E.

    Barbara Pergolizzi, Cristina Panuzzo, M. Shahzad Ali, Marco Lo Iacono, Chiara Levra Levron, Luca Ponzone, Marta Prelli, Daniela Cilloni, Enzo Calautti, Salvatore Bozzaro, and Enrico Bracco Mammalian, or mechanistic, target of rapamycin complex 2 (mTORC2) regulates a variety of vital cellular processes, and its aberrant functioning is often associated with various diseases. Rictor is a peculiar and distinguishing mTORC2 component playing a pivotal role in controlling its assembly and activity. Among extant organisms, Rictor is conserved from unicellular eukaryotes to metazoans. We replaced two distinct, but conserved, glycine residues in both the Dictyostelium piaA gene and its human ortholog, RICTOR. The two conserved residues are spaced ~50 amino acids apart, and both are embedded within a conserved region falling in between the Ras-GEFN2 and Rictor­_V domains. The effects of point mutations on the mTORC2 activity and integrity were assessed by biochemical and functional assays. In both cases, these equivalent point mutations in the mammalian RICTOR and Dictyostelium piaA gene impaired mTORC2 activity and integrity. Our data indicate that the two glycine residues are essential for the maintenance of mTORC2 activity and integrity in organisms that appear to be distantly related, suggesting that they have a evolutionarily conserved role in the assembly and proper mTORC2 functioning.

    更新日期:2019-11-29
  • Spatial integration of mechanical forces by {alpha}-actinin establishes actin network symmetry
    J. Cell Sci. (IF 4.401) Pub Date : 2019-11-14
    Senger, F., Pitaval, A., Ennomani, H., Kurzawa, L., Blanchoin, L., Thery, M.

    Fabrice Senger, Amandine Pitaval, Hajer Ennomani, Laetitia Kurzawa, Laurent Blanchoin, and Manuel Thery Cell and tissue morphogenesis depend on the production and spatial organization of tensional forces in the actin cytoskeleton. Actin network architecture is made of distinct modules characterized by specific filament organizations. The assembly of these modules are well described, but their integration in a cellular network is less understood. Here, we investigated the mechanism regulating the interplay between network architecture and the geometry of the extracellular environment of the cell. We found that α-actinin, a filament crosslinker, is essential for network symmetry to be consistent with extracellular microenvironment symmetry. It is required for the interconnection of transverse arcs with radial fibres to ensure an appropriate balance between forces at cell adhesions and across the actin network. Furthermore, this connectivity appeared necessary for the ability of the cell to integrate and to adapt to complex patterns of extracellular cues as they migrate. Our study has unveiled a role of actin filament crosslinking in the spatial integration of mechanical forces that ensures the adaptation of intracellular symmetry axes in accordance with the geometry of extracellular cues. This article has an associated First Person interview with the first author of the paper.

    更新日期:2019-11-29
  • Nutrient regulation of mTORC1 at a glance
    J. Cell Sci. (IF 4.401) Pub Date : 2019-11-13
    Condon, K. J., Sabatini, D. M.

    Kendall J. Condon and David M. Sabatini The mechanistic target of rapamycin (mTOR) signaling pathway coordinates environmental and intracellular cues to control eukaryotic cell growth. As a pivot point between anabolic and catabolic processes, mTOR complex 1 (mTORC1) signaling has established roles in regulating metabolism, translation and autophagy. Hyperactivity of the mTOR pathway is associated with numerous human diseases, including diabetes, cancer and epilepsy. Pharmacological inhibition of the mTOR pathway can extend lifespan in a variety of model organisms. Given its broad control of essential cellular processes and clear relevance to human health, there is extensive interest in elucidating how upstream inputs regulate mTORC1 activation. In this Cell Science at a Glance article and accompanying poster, we summarize our understanding of how extracellular and intracellular signals feed into the mTOR pathway, how the lysosome acts as an mTOR signaling hub, and how downstream signaling controls autophagy and lysosome biogenesis.

    更新日期:2019-11-14
  • Rap1 and membrane lipids cooperatively recruit talin to trigger integrin activation
    J. Cell Sci. (IF 4.401) Pub Date : 2019-11-01
    Bromberger, T., Zhu, L., Klapproth, S., Qin, J., Moser, M.

    Thomas Bromberger, Liang Zhu, Sarah Klapproth, Jun Qin, and Markus Moser Recruitment and tethering of talin to the plasma membrane initiate the process of integrin activation. Multiple factors including the Rap1 proteins, RIAM (also known as APBB1IP) and PIP2 bind talin proteins and have been proposed to regulate these processes, but not systematically analyzed. By expressing specific talin mutants into talin-null fibroblasts, we show that binding of the talin F0 domain to Rap1 synergizes with membrane lipid binding of the talin F2 domain during talin membrane targeting and integrin activation, whereas the interaction of the talin rod with RIAM was dispensable. We also characterized a second Rap1-binding site within the talin F1 domain by detailed NMR analysis. Interestingly, while talin F1 exhibited significantly weaker Rap1-binding affinity than talin F0, expression of a talin F1 Rap1-binding mutant inhibited cell adhesion, spreading, talin recruitment and integrin activation similarly to the talin F0 Rap1-binding mutant. Moreover, the defects became significantly stronger when both Rap1-binding sites were mutated. In conclusion, our data suggest a model in which cooperative binding of Rap1 to the talin F0 and F1 domains synergizes with membrane PIP2 binding to spatiotemporally position and activate talins to regulate integrin activity.

    更新日期:2019-11-14
  • Reversible solidification of fission yeast cytoplasm after prolonged nutrient starvation
    J. Cell Sci. (IF 4.401) Pub Date : 2019-11-08
    Heimlicher, M. B., Bächler, M., Liu, M., Ibeneche-Nnewihe, C., Florin, E.-L., Hoenger, A., Brunner, D.

    Maria B. Heimlicher, Mirjam Bächler, Minghua Liu, Chieze Ibeneche-Nnewihe, Ernst-Ludwig Florin, Andreas Hoenger, and Damian Brunner Cells depend on a highly ordered organisation of their content and must develop strategies to maintain the anisotropic distribution of organelles during periods of nutrient shortage. One of these strategies is to solidify the cytoplasm, which was observed in bacteria and yeast cells with acutely interrupted energy production. Here, we describe a different type of cytoplasm solidification fission yeast cells switch to, after having run out of nutrients during multiple days in culture. It provides the most profound reversible cytoplasmic solidification of yeast cells described to date. Our data exclude the previously proposed mechanisms for cytoplasm solidification in yeasts and suggest a mechanism that immobilises cellular components in a size-dependent manner. We provide experimental evidence that, in addition to time, cells use intrinsic nutrients and energy sources to reach this state. Such cytoplasmic solidification may provide a robust means to protect cellular architecture in dormant cells.

    更新日期:2019-11-14
  • SIRT2 deacetylates GRASP55 to facilitate post-mitotic Golgi assembly
    J. Cell Sci. (IF 4.401) Pub Date : 2019-11-01
    Zhang, X., Brachner, A., Kukolj, E., Slade, D., Wang, Y.

    Xiaoyan Zhang, Andreas Brachner, Eva Kukolj, Dea Slade, and Yanzhuang Wang Sirtuin 2 (SIRT2) is an NAD-dependent sirtuin deacetylase that regulates microtubule and chromatin dynamics, gene expression and cell cycle progression, as well as nuclear envelope reassembly. Recent proteomic analyses have identified Golgi proteins as SIRT2 interactors, indicating that SIRT2 may also play a role in Golgi structure formation. Here, we show that SIRT2 depletion causes Golgi fragmentation and impairs Golgi reassembly at the end of mitosis. SIRT2 interacts with the Golgi reassembly stacking protein GRASP55 (also known as GORASP2) in mitosis when GRASP55 is highly acetylated on K50. Expression of wild-type and the K50R acetylation-deficient mutant of GRASP55, but not the K50Q acetylation-mimetic mutant, in GRASP55 and GRASP65 (also known as GORASP1) double-knockout cells, rescued the Golgi structure and post-mitotic Golgi reassembly. Acetylation-deficient GRASP55 exhibited a higher self-interaction efficiency, a property required for Golgi structure formation. These results demonstrate that SIRT2 regulates Golgi structure by modulating GRASP55 acetylation levels.

    更新日期:2019-11-14
  • Cul3 regulates cyclin E1 protein abundance via a degron located within the N-terminal region of cyclin E
    J. Cell Sci. (IF 4.401) Pub Date : 2019-11-06
    Davidge, B., Rebola, K. G. d. O., Agbor, L. N., Sigmund, C. D., Singer, J. D.

    Brittney Davidge, Katia Graziella de Oliveira Rebola, Larry N. Agbor, Curt D. Sigmund, and Jeffrey D. Singer Cyclin E and its binding partner Cdk2 control the G1/S transition in mammalian cells. Increased levels of cyclin E are found in some cancers. Additionally, proteolytic removal of the cyclin E N-terminus occurs in some cancers and is associated with increased cyclin E–Cdk2 activity and poor clinical prognosis. Cyclin E levels are tightly regulated and controlled in part through ubiquitin-mediated degradation initiated by one of two E3 ligases, Cul1 and Cul3. Cul1 ubiquitylates phosphorylated cyclin E, but the mechanism through which Cul3 ubiquitylates cyclin E is poorly understood. In experiments to ascertain how Cul3 mediates cyclin E destruction, we identified a degron on cyclin E that Cul3 targets for ubiquitylation. Recognition of the degron and binding of Cul3 does not require a BTB domain-containing adaptor protein. Additionally, this degron is lacking in N-terminally truncated cyclin E. Our results describe a mechanism whereby N-terminally truncated cyclin E can avoid the Cul3-mediated degradation pathway. This mechanism helps to explain the increased activity that is associated with the truncated cyclin E variants that occurs in some cancers.

    更新日期:2019-11-14
  • P-cadherin-induced decorin secretion is required for collagen fiber alignment and directional collective cell migration
    J. Cell Sci. (IF 4.401) Pub Date : 2019-11-08
    Le Borgne-Rochet, M., Angevin, L., Bazellieres, E., Ordas, L., Comunale, F., Denisov, E. V., Tashireva, L. A., Perelmuter, V. M., Bieche, I., Vacher, S., Plutoni, C., Seveno, M., Bodin, S., Gauthier-Rouviere, C.

    Maïlys Le Borgne-Rochet, Lucie Angevin, Elsa Bazellieres, Laura Ordas, Franck Comunale, Evgeny V. Denisov, Lubov A. Tashireva, Vladimir M. Perelmuter, Ivan Bieche, Sophie Vacher, Cedric Plutoni, Martial Seveno, Stephane Bodin, and Cecile Gauthier-Rouviere Directional collective cell migration (DCCM) is crucial for morphogenesis and cancer metastasis. P-cadherin (also known as CDH3), which is a cell–cell adhesion protein expressed in carcinoma and aggressive sarcoma cells and associated with poor prognosis, is a major DCCM regulator. However, it is unclear how P-cadherin-mediated mechanical coupling between migrating cells influences force transmission to the extracellular matrix (ECM). Here, we found that decorin, a small proteoglycan that binds to and organizes collagen fibers, is specifically expressed and secreted upon P-cadherin, but not E- and R-cadherin (also known as CDH1 and CDH4, respectively) expression. Through cell biological and biophysical approaches, we demonstrated that decorin is required for P-cadherin-mediated DCCM and collagen fiber orientation in the migration direction in 2D and 3D matrices. Moreover, P-cadherin, through decorin-mediated collagen fiber reorientation, promotes the activation of β1 integrin and of the β-Pix (ARHGEF7)/CDC42 axis, which increases traction forces, allowing DCCM. Our results identify a novel P-cadherin-mediated mechanism to promote DCCM through ECM remodeling and ECM-guided cell migration.

    更新日期:2019-11-14
  • Cell cycle-independent furrowing triggered by phosphomimetic mutations of the INCENP STD motif requires Plk1
    J. Cell Sci. (IF 4.401) Pub Date : 2019-11-06
    Papini, D., Fant, X., Ogawa, H., Desban, N., Samejima, K., Feizbakhsh, O., Askin, B., Ly, T., Earnshaw, W. C., Ruchaud, S.

    Diana Papini, Xavier Fant, Hiromi Ogawa, Nathalie Desban, Kumiko Samejima, Omid Feizbakhsh, Bilge Askin, Tony Ly, William C. Earnshaw, and Sandrine Ruchaud Timely and precise control of Aurora B kinase, the chromosomal passenger complex (CPC) catalytic subunit, is essential for accurate chromosome segregation and cytokinesis. Post-translational modifications of CPC subunits are directly involved in controlling Aurora B activity. Here, we identified a highly conserved acidic STD-rich motif of INCENP that is phosphorylated during mitosis in vivo and by Plk1 in vitro and is involved in controlling Aurora B activity. By using an INCENP conditional-knockout cell line, we show that impairing the phosphorylation status of this region disrupts chromosome congression and induces cytokinesis failure. In contrast, mimicking constitutive phosphorylation not only rescues cytokinesis but also induces ectopic furrows and contractile ring formation in a Plk1- and ROCK1-dependent manner independent of cell cycle and microtubule status. Our experiments identify the phospho-regulation of the INCENP STD motif as a novel mechanism that is key for chromosome alignment and cytokinesis. This article has an associated First Person interview with the first author of the paper.

    更新日期:2019-11-14
  • Adaptor protein complexes and disease at a glance
    J. Cell Sci. (IF 4.401) Pub Date : 2019-10-21
    Sanger, A., Hirst, J., Davies, A. K., Robinson, M. S.

    Anneri Sanger, Jennifer Hirst, Alexandra K. Davies, and Margaret S. Robinson Adaptor protein (AP) complexes are heterotetramers that select cargo for inclusion into transport vesicles. Five AP complexes (AP-1 to AP-5) have been described, each with a distinct localisation and function. Furthermore, patients with a range of disorders, particularly involving the nervous system, have now been identified with mutations in each of the AP complexes. In many cases this has been correlated with aberrantly localised membrane proteins. In this Cell Science at a Glance article and the accompanying poster, we summarize what is known about the five AP complexes and discuss how this helps to explain the clinical features of the different genetic disorders.

    更新日期:2019-11-01
  • Identification of matrix physicochemical properties required for renal epithelial cell tubulogenesis by using synthetic hydrogels
    J. Cell Sci. (IF 4.401) Pub Date : 2019-10-21
    Cruz-Acuna, R., Mulero-Russe, A., Clark, A. Y., Zent, R., Garcia, A. J.

    Ricardo Cruz-Acuna, Adriana Mulero-Russe, Amy Y. Clark, Roy Zent, and Andres J. Garcia Synthetic hydrogels with controlled physicochemical matrix properties serve as powerful in vitro tools to dissect cell–extracellular matrix (ECM) interactions that regulate epithelial morphogenesis in 3D microenvironments. In addition, these fully defined matrices overcome the lot-to-lot variability of naturally derived materials and have provided insights into the formation of rudimentary epithelial organs. Therefore, we engineered a fully defined synthetic hydrogel with independent control over proteolytic degradation, mechanical properties, and adhesive ligand type and density to study the impact of ECM properties on epithelial tubulogenesis for inner medullary collecting duct (IMCD) cells. Protease sensitivity of the synthetic material for membrane-type matrix metalloproteinase-1 (MT1-MMP, also known as MMP14) was required for tubulogenesis. Additionally, a defined range of matrix elasticity and presentation of RGD adhesive peptide at a threshold level of 2 mM ligand density were required for epithelial tubulogenesis. Finally, we demonstrated that the engineered hydrogel supported organization of epithelial tubules with a lumen and secreted laminin. This synthetic hydrogel serves as a platform that supports epithelial tubular morphogenetic programs and can be tuned to identify ECM biophysical and biochemical properties required for epithelial tubulogenesis.

    更新日期:2019-11-01
  • Invadopodia-mediated ECM degradation is enhanced in the G1 phase of the cell cycle
    J. Cell Sci. (IF 4.401) Pub Date : 2019-10-18
    Bayarmagnai, B., Perrin, L., Esmaeili Pourfarhangi, K., Grana, X., Tüzel, E., Gligorijevic, B.

    Battuya Bayarmagnai, Louisiane Perrin, Kamyar Esmaeili Pourfarhangi, Xavier Grana, Erkan Tüzel, and Bojana Gligorijevic The process of tumor cell invasion and metastasis includes assembly of invadopodia, protrusions capable of degrading the extracellular matrix (ECM). The effect of cell cycle progression on invadopodia has not been elucidated. In this study, by using invadopodia and cell cycle fluorescent markers, we show in 2D and 3D cultures, as well as in vivo, that breast carcinoma cells assemble invadopodia and invade into the surrounding ECM preferentially during the G1 phase. The expression (MT1-MMP, also known as MMP14, and cortactin) and localization (Tks5; also known as SH3PXD2A) of invadopodia components are elevated in G1 phase, and cells synchronized in G1 phase exhibit significantly higher ECM degradation compared to the cells synchronized in S phase. The cyclin-dependent kinase inhibitor (CKI) p27kip1 (also known as CDKN1B) localizes to the sites of invadopodia assembly. Overexpression and stable knockdown of p27kip1 lead to contrasting effects on invadopodia turnover and ECM degradation. Taken together, these findings suggest that expression of invadopodia components, as well as invadopodia function, are linked to cell cycle progression, and that invadopodia are controlled by cell cycle regulators. Our results caution that this coordination between invasion and cell cycle must be considered when designing effective chemotherapies.

    更新日期:2019-11-01
  • Perturbed mitochondria-ER contacts in live neurons that model the amyloid pathology of Alzheimer's disease
    J. Cell Sci. (IF 4.401) Pub Date : 2019-10-22
    Martino Adami, P. V., Nichtova, Z., Weaver, D. B., Bartok, A., Wisniewski, T., Jones, D. R., Do Carmo, S., Castano, E. M., Cuello, A. C., Hajnoczky, G., Morelli, L.

    Pamela V. Martino Adami, Zuzana Nichtova, David B. Weaver, Adam Bartok, Thomas Wisniewski, Drew R. Jones, Sonia Do Carmo, Eduardo M. Castano, A. Claudio Cuello, György Hajnoczky, and Laura Morelli The use of fixed fibroblasts from familial and sporadic Alzheimer's disease patients has previously indicated an upregulation of mitochondria–ER contacts (MERCs) as a hallmark of Alzheimer's disease. Despite its potential significance, the relevance of these results is limited because they were not extended to live neurons. Here we performed a dynamic in vivo analysis of MERCs in hippocampal neurons from McGill-R-Thy1-APP transgenic rats, a model of Alzheimer's disease-like amyloid pathology. Live FRET imaging of neurons from transgenic rats revealed perturbed ‘lipid-MERCs’ (gap width <10 nm), while ‘Ca2+-MERCs’ (10–20 nm gap width) were unchanged. In situ TEM showed no significant differences in the lipid-MERCs:total MERCs or lipid-MERCs:mitochondria ratios; however, the average length of lipid-MERCs was significantly decreased in neurons from transgenic rats as compared to controls. In accordance with FRET results, untargeted lipidomics showed significant decreases in levels of 12 lipids and bioenergetic analysis revealed respiratory dysfunction of mitochondria from transgenic rats. Thus, our results reveal changes in MERC structures coupled with impaired mitochondrial functions in Alzheimer's disease-related neurons. This article has an associated First Person interview with the first author of the paper.

    更新日期:2019-11-01
  • FKBP52 regulates TRPC3-dependent Ca2+ signals and the hypertrophic growth of cardiomyocyte cultures
    J. Cell Sci. (IF 4.401) Pub Date : 2019-10-23
    Bandleon, S., Strunz, P. P., Pickel, S., Tiapko, O., Cellini, A., Miranda-Laferte, E., Eder-Negrin, P.

    Sandra Bandleon, Patrick P. Strunz, Simone Pickel, Oleksandra Tiapko, Antonella Cellini, Erick Miranda-Laferte, and Petra Eder-Negrin The transient receptor potential (TRP; C-classical, TRPC) channel TRPC3 allows a cation (Na+/Ca2+) influx that is favored by the stimulation of Gq protein-coupled receptors (GPCRs). An enhanced TRPC3 activity is related to adverse effects, including pathological hypertrophy in chronic cardiac disease states. In the present study, we identified FK506-binding protein 52 (FKBP52, also known as FKBP4) as a novel interaction partner of TRPC3 in the heart. FKBP52 was recovered from a cardiac cDNA library by a C-terminal TRPC3 fragment (amino acids 742–848) in a yeast two-hybrid screen. Downregulation of FKBP52 promoted a TRPC3-dependent hypertrophic response in neonatal rat cardiomyocytes (NRCs). A similar effect was achieved by overexpressing peptidyl-prolyl isomerase (PPIase)-deficient FKBP52 mutants. Mechanistically, expression of the FKBP52 truncation mutants elevated TRPC3-mediated currents and Ca2+ fluxes, and the activation of calcineurin and the nuclear factor of activated T-cells in NRCs. Our data demonstrate that FKBP52 associates with TRPC3 via an as-yet-undescribed binding site in the C-terminus of TRPC3 and modulates TRPC3-dependent Ca2+ signals in a PPIase-dependent manner. This functional interaction might be crucial for limiting TRPC3-dependent signaling during chronic hypertrophic stimulation.

    更新日期:2019-11-01
  • Mannose glycosylation is an integral step for NIS localization and function in human breast cancer cells
    J. Cell Sci. (IF 4.401) Pub Date : 2019-10-23
    Rathod, M., Chatterjee, S., Dutta, S., Kalraiya, R., Bhattacharyya, D., De, A.

    Maitreyi Rathod, Sushmita Chatterjee, Shruti Dutta, Rajiv Kalraiya, Dibyendu Bhattacharyya, and Abhijit De Chasing an intriguing biological question on the disparity of sodium iodide symporter (NIS, officially known as SLC5A5) expression and function in the clinical scenario of breast cancer, this study addresses key molecular defects involved. NIS in cancer patients has primarily been recorded to be a cytoplasmic protein, thus limiting the scope for targeted radio-iodine therapy. We developed NIS transgene-overexpressing MCF-7 breast cancer cells, and found a few clonal derivatives that show predominant expression of NIS in the plasma membrane. The majority of clones, however, showed cytosolic NIS expression over long passages. Cells expressing membranous NIS show unperturbed dynamic trafficking of NIS through secretory pathway organelles when compared to cells expressing cytoplasmic NIS or to parental cells. Further, treatment of cells expressing membranous NIS with specific glycosylation inhibitors highlighted the importance of inherent glycosylation processing and an 84 gene signature glycosylation RT-Profiler array revealed that clones expressing NIS in their membrane cluster separately compared to the other cells. We further confirm a role of three differentially expressed genes, i.e. MAN1B1, MAN1A1 and MAN2A1, in regulating NIS localization by RNA interference. Thus, this study shows the important role of mannosidase in N-glycosylation processing in order to correctly traffic NIS to the plasma membrane in breast cancer cells. This article has an associated First Person interview with the first author of the paper.

    更新日期:2019-11-01
  • Tox4 modulates cell fate reprogramming
    J. Cell Sci. (IF 4.401) Pub Date : 2019-10-22
    Vanheer, L., Song, J., De Geest, N., Janiszewski, A., Talon, I., Provenzano, C., Oh, T., Chappell, J., Pasque, V.

    Lotte Vanheer, Juan Song, Natalie De Geest, Adrian Janiszewski, Irene Talon, Caterina Provenzano, Taeho Oh, Joel Chappell, and Vincent Pasque Reprogramming to induced pluripotency induces the switch of somatic cell identity to induced pluripotent stem cells (iPSCs). However, the mediators and mechanisms of reprogramming remain largely unclear. To elucidate the mediators and mechanisms of reprogramming, we used a siRNA-mediated knockdown approach for selected candidate genes during the conversion of somatic cells into iPSCs. We identified Tox4 as a novel factor that modulates cell fate through an assay that determined the efficiency of iPSC reprogramming. We found that Tox4 is needed early in reprogramming to efficiently generate early reprogramming intermediates, irrespective of the reprogramming conditions used. Tox4 enables proper exogenous reprogramming factor expression, and the closing and opening of putative somatic and pluripotency enhancers early during reprogramming, respectively. We show that the TOX4 protein assembles into a high molecular form. Moreover, Tox4 is also required for the efficient conversion of fibroblasts towards the neuronal fate, suggesting a broader role of Tox4 in modulating cell fate. Our study reveals Tox4 as a novel transcriptional modulator of cell fate that mediates reprogramming from the somatic state to the pluripotent and neuronal fate. This article has an associated First Person interview with the first author of the paper.

    更新日期:2019-11-01
  • Annexin A5 regulates surface {alpha}v{beta}5 integrin for retinal clearance phagocytosis
    J. Cell Sci. (IF 4.401) Pub Date : 2019-10-16
    Yu, C., Munoz, L. E., Mallavarapu, M., Herrmann, M., Finnemann, S. C.

    Chen Yu, Luis E. Munoz, Mallika Mallavarapu, Martin Herrmann, and Silvia C. Finnemann Diurnal clearance phagocytosis by the retinal pigment epithelium (RPE) is a conserved efferocytosis process whose binding step is mediated by αvβ5 integrin receptors. Two related annexins, A5 (ANXA5) and A6 (ANXA6), share an αvβ5 integrin-binding motif. Here, we report that ANXA5, but not ANXA6, regulates the binding capacity for spent photoreceptor outer segment fragments or apoptotic cells by fibroblasts and RPE. Similar to αvβ5-deficient RPE, ANXA5–/– RPE in vivo lacks the diurnal burst of phagocytosis that follows photoreceptor shedding in wild-type retina. Increasing ANXA5 in cells lacking αvβ5 or increasing αvβ5 in cells lacking ANXA5 does not affect particle binding. Association of cytosolic ANXA5 and αvβ5 integrin in RPE in culture and in vivo further supports their functional interdependence. Silencing ANXA5 is sufficient to reduce levels of αvβ5 receptors at the apical phagocytic surface of RPE cells. The effect of ANXA5 on surface αvβ5 and on particle binding requires the C-terminal ANXA5 annexin repeat but not its unique N-terminus. These results identify a novel role for ANXA5 specifically in the recognition and binding step of clearance phagocytosis, which is essential to retinal physiology. This article has an associated First Person interview with the first author of the paper.

    更新日期:2019-11-01
  • VLA-4 phosphorylation during tumor and immune cell migration relies on its coupling to VEGFR2 and CXCR4 by syndecan-1
    J. Cell Sci. (IF 4.401) Pub Date : 2019-10-28
    Jung, O., Beauvais, D. M., Adams, K. M., Rapraeger, A. C.

    Oisun Jung, DeannaLee M. Beauvais, Kristin M. Adams, and Alan C. Rapraeger When targeted by the tumor-promoting enzyme heparanase, cleaved and shed syndecan-1 (Sdc1) then couples VEGFR2 (also known as KDR) to VLA-4, activating VEGFR2 and the directed migration of myeloma cells. But how VEGFR2 activates VLA-4-mediated motility has remained unknown. We now report that VEGFR2 causes PKA-mediated phosphorylation of VLA-4 on S988, an event known to stimulate tumor metastasis while suppressing cytotoxic immune cells. A key partner in this mechanism is the chemokine receptor CXCR4, a well-known mediator of cell motility in response to gradients of the chemokine SDF-1 (also known as CXCL12). The entire machinery necessary to phosphorylate VLA-4, consisting of CXCR4, AC7 (also known as ADCY7) and PKA, is constitutively associated with VEGFR2 and is localized to the integrin by Sdc1. VEGFR2 carries out the novel phosphorylation of Y135 within the DRY microswitch of CXCR4, sequentially activating Gαiβ, AC7 and PKA, which phosphorylates S988 on the integrin. This mechanism is blocked by a syndecan-mimetic peptide (SSTNVEGFR2), which, by preventing VEGFR2 linkage to VLA-4, arrests tumor cell migration that depends on VLA-4 phosphorylation and stimulates the LFA-1-mediated migration of cytotoxic leukocytes.

    更新日期:2019-11-01
  • An autism-associated mutation in GluN2B prevents NMDA receptor trafficking and interferes with dendrite growth
    J. Cell Sci. (IF 4.401) Pub Date : 2019-10-18
    Sceniak, M. P., Fedder, K. N., Wang, Q., Droubi, S., Babcock, K., Patwardhan, S., Wright-Zornes, J., Pham, L., Sabo, S. L.

    Michael P. Sceniak, Karlie N. Fedder, Qian Wang, Sammy Droubi, Katie Babcock, Sagar Patwardhan, Jazmin Wright-Zornes, Lucynda Pham, and Shasta L. Sabo Autism spectrum disorders (ASDs) are neurodevelopmental disorders with multiple genetic associations. Analysis of de novo mutations identified GRIN2B, which encodes the GluN2B subunit of NMDA receptors, as a gene linked to ASDs with high probability. However, the mechanisms by which GRIN2B mutations contribute to ASD pathophysiology are not understood. Here, we investigated the cellular phenotypes induced by a human mutation that is predicted to truncate GluN2B within the extracellular loop. This mutation abolished NMDA-dependent Ca2+ influx. Mutant GluN2B co-assembled with GluN1 but was not trafficked to the cell surface or dendrites. When mutant GluN2B was expressed in developing cortical neurons, dendrites appeared underdeveloped, with shorter and fewer branches, while spine density was unaffected. Mutant dendritic arbors were often dysmorphic, displaying abnormal filopodial-like structures. Interestingly, dendrite maldevelopment appeared when mutant GluN2B was expressed on a wild-type background, reflecting the disease given that individuals are heterozygous for GRIN2B mutations. Restoring the fourth transmembrane domain and cytoplasmic tail did not rescue the phenotypes. Finally, abnormal development was not accompanied by reduced mTOR signaling. These data suggest that mutations in GluN2B contribute to ASD pathogenesis by disrupting dendrite development.

    更新日期:2019-11-01
  • The Parkinson's disease gene PINK1 activates Akt via PINK1 kinase-dependent regulation of the phospholipid PI(3,4,5)P3
    J. Cell Sci. (IF 4.401) Pub Date : 2019-10-22
    Furlong, R. M., Lindsay, A., Anderson, K. E., Hawkins, P. T., Sullivan, A. M., O'Neill, C.

    Rachel M. Furlong, Andrew Lindsay, Karen E. Anderson, Phillip T. Hawkins, Aideen M. Sullivan, and Cora O'Neill Akt signalling is central to cell survival, metabolism, protein and lipid homeostasis, and is impaired in Parkinson's disease (PD). Akt activation is reduced in the brain in PD, and by many PD-causing genes, including PINK1. This study investigated the mechanisms by which PINK1 regulates Akt signalling. Our results reveal for the first time that PINK1 constitutively activates Akt in a PINK1-kinase dependent manner in the absence of growth factors, and enhances Akt activation in normal growth medium. In PINK1-modified MEFs, agonist-induced Akt signalling failed in the absence of PINK1, due to PINK1 kinase-dependent increases in PI(3,4,5)P3 at both plasma membrane and Golgi being significantly impaired. In the absence of PINK1, PI(3,4,5)P3 levels did not increase in the Golgi, and there was significant Golgi fragmentation, a recognised characteristic of PD neuropathology. PINK1 kinase activity protected the Golgi from fragmentation in an Akt-dependent fashion. This study demonstrates a new role for PINK1 as a primary upstream activator of Akt via PINK1 kinase-dependent regulation of its primary activator PI(3,4,5)P3, providing novel mechanistic information on how loss of PINK1 impairs Akt signalling in PD. This article has an associated First Person interview with the first author of the paper.

    更新日期:2019-11-01
  • TGF{beta}-induced fibroblast activation requires persistent and targeted HDAC-mediated gene repression
    J. Cell Sci. (IF 4.401) Pub Date : 2019-10-18
    Jones, D. L., Haak, A. J., Caporarello, N., Choi, K. M., Ye, Z., Yan, H., Varelas, X., Ordog, T., Ligresti, G., Tschumperlin, D. J.

    Dakota L. Jones, Andrew J. Haak, Nunzia Caporarello, Kyoung M. Choi, Zhenqing Ye, Huihuang Yan, Xaralabos Varelas, Tamas Ordog, Giovanni Ligresti, and Daniel J. Tschumperlin Tissue fibrosis is a chronic disease driven by persistent fibroblast activation that has recently been linked to epigenetic modifications. Here, we screened a small library of epigenetic small-molecule modulators to identify compounds capable of inhibiting or reversing TGFβ-mediated fibroblast activation. We identified pracinostat, an HDAC inhibitor, as a potent attenuator of lung fibroblast activation and confirmed its efficacy in patient-derived fibroblasts isolated from fibrotic lung tissue. Mechanistically, we found that HDAC-dependent transcriptional repression was an early and essential event in TGFβ-mediated fibroblast activation. Treatment of lung fibroblasts with pracinostat broadly attenuated TGFβ-mediated epigenetic repression and promoted fibroblast quiescence. We confirmed a specific role for HDAC-dependent histone deacetylation in the promoter region of the anti-fibrotic gene PPARGC1A (PGC1α) in response to TGFβ stimulation. Finally, we identified HDAC7 as a key factor whose siRNA-mediated knockdown attenuates fibroblast activation without altering global histone acetylation. Together, these results provide novel mechanistic insight into the essential role HDACs play in TGFβ-mediated fibroblast activation via targeted gene repression.

    更新日期:2019-11-01
  • Inhibiting SUMO1-mediated SUMOylation induces autophagy-mediated cancer cell death and reduces tumour cell invasion via RAC1
    J. Cell Sci. (IF 4.401) Pub Date : 2019-10-22
    Lorente, M., Garcia-Casas, A., Salvador, N., Martinez-Lopez, A., Gabicagogeascoa, E., Velasco, G., Lopez-Palomar, L., Castillo-Lluva, S.

    Mar Lorente, Ana Garcia-Casas, Nelida Salvador, Angelica Martinez-Lopez, Estibaliz Gabicagogeascoa, Guillermo Velasco, Lucia Lopez-Palomar, and Sonia Castillo-Lluva Post-translational modifications directly control protein activity and, thus, they represent an important means to regulate the responses of cells to different stimuli. Protein SUMOylation has recently been recognised as one such modification, and it has been associated with various diseases, including different types of cancer. However, the precise way that changes in SUMOylation influence the tumorigenic properties of cells remains to be fully clarified. Here, we show that blocking the SUMO pathway by depleting SUMO1 and UBC9, or by exposure to ginkgolic acid C15:1 or 2-D08 (two different SUMOylation inhibitors), induces cell death, also inhibiting the invasiveness of tumour cells. Indeed, diminishing the formation of SUMO1 complexes induces autophagy-mediated cancer cell death through increasing the expression of Tribbles pseudokinase 3 (TRIB3). Moreover, we found that blocking the SUMO pathway inhibits tumour cell invasion by decreasing RAC1 SUMOylation. These findings shed new light on the mechanisms by which SUMO1 modifications regulate the survival, and the migratory and invasive capacity of tumour cells, potentially establishing the bases to develop novel anti-cancer treatments based on the inhibition of SUMOylation.

    更新日期:2019-11-01
  • Drosophila emerins control LINC complex localization and transcription to regulate myonuclear position
    J. Cell Sci. (IF 4.401) Pub Date : 2019-10-18
    Mandigo, T. R., Turcich, B. D., Anderson, A. J., Hussey, M. R., Folker, E. S.

    Torrey R. Mandigo, Blake D. Turcich, Alyssa J. Anderson, Michael R. Hussey, and Eric S. Folker Mispositioned nuclei are a hallmark of skeletal muscle disease. Many of the genes that are linked to Emery–Dreifuss muscular dystrophy (EDMD) encode proteins that are critical for nuclear movement in various cells, suggesting that disruptions in nuclear movement and position may contribute to disease progression. However, how these genes are coordinated to move nuclei is not known. Here, we focussed on two different emerin proteins in Drosophila, Bocksbeutel and Otefin, and their effects on nuclear movement. Although nuclear position was dependent on both, elimination of either Bocksbeutel or Otefin produced distinct phenotypes that were based in differential effects on the KASH-domain protein Klarsicht. Specifically, loss of Bocksbeutel reduced Klarsicht localization to the nucleus and resulted in a disruption in nuclear separation. Loss of Otefin increased the transcription of Klarsicht and led to premature separation of nuclei and their positioning closer to the edge of the muscle. Consistent with opposing functions, nuclear position is normal in otefin; bocksbeutel double mutants. These data indicate emerin-dependent regulation of Klarsicht levels in the nuclear envelope is a critical determinant of nuclear position.

    更新日期:2019-11-01
  • Cyclin-dependent kinase 1-mediated AMPK phosphorylation regulates chromosome alignment and mitotic progression
    J. Cell Sci. (IF 4.401) Pub Date : 2019-10-28
    Stauffer, S., Zeng, Y., Santos, M., Zhou, J., Chen, Y., Dong, J.

    Seth Stauffer, Yongji Zeng, Montserrat Santos, Jiuli Zhou, Yuanhong Chen, and Jixin Dong AMP-activated protein kinase (AMPK), a heterotrimeric serine/threonine kinase and cellular metabolic sensor, has been found to regulate cell cycle checkpoints in cancer cells in response to energetic stress, to harmonize proliferation with energy availability. Despite AMPK's emergent association with the cell cycle, it still has not been fully delineated how AMPK is regulated by upstream signaling pathways during mitosis. We report, for the first time, direct CDK1 phosphorylation of both the catalytic α1 and α2 subunits, as well as the β1 regulatory subunit, of AMPK in mitosis. We found that AMPK-knockout U2OS osteosarcoma cells have reduced mitotic indexes and that CDK1 phosphorylation-null AMPK is unable to rescue the phenotype, demonstrating a role for CDK1 regulation of mitotic entry through AMPK. Our results also denote a vital role for AMPK in promoting proper chromosomal alignment, as loss of AMPK activity leads to misaligned chromosomes and concomitant metaphase delay. Importantly, AMPK expression and activity was found to be critical for paclitaxel chemosensitivity in breast cancer cells and positively correlated with relapse-free survival in systemically treated breast cancer patients.

    更新日期:2019-11-01
  • The lncRNA hsr{omega} regulates arginine dimethylation of human FUS to cause its proteasomal degradation in Drosophila
    J. Cell Sci. (IF 4.401) Pub Date : 2019-10-23
    Lo Piccolo, L., Mochizuki, H., Nagai, Y.

    Luca Lo Piccolo, Hideki Mochizuki, and Yoshitaka Nagai Long non-coding RNAs (lncRNAs) have structural and regulatory effects on RNA-binding proteins (RBPs). However, the mechanisms by which lncRNAs regulate the neurodegenerative-causative RBP like FUS protein remain poorly understood. Here, we show that knockdown of the Drosophila lncRNA hsr causes a shift in the methylation status of human FUS from mono- (MMA) to di-methylated (DMA) arginine via upregulation of the arginine methyltransferase 5 (PRMT5, known as ART5 in flies). We found this novel regulatory role to be critical for FUS toxicity since the PRMT5-dependent dimethylation of FUS is required for its proteasomal degradation and causes a reduction of high levels of FUS. Moreover, we show that an increase of FUS causes a decline of both PRMT1 (known as ART1 in flies) and PRMT5 transcripts, leading to an accumulation of neurotoxic MMA-FUS. Therefore, overexpression of either PRMT1 or PRMT5 is able to rescue the FUS toxicity. These results highlight a novel role of lncRNAs in post-translation modification (PTM) of FUS and suggest a causal relationship between lncRNAs and dysfunctional PRMTs in the pathogenesis of FUSopathies.

    更新日期:2019-11-01
  • Molecular regulation of Snai2 in development and disease.
    J. Cell Sci. (IF 4.401) Pub Date : null
    Wenhui Zhou,Kayla M Gross,Charlotte Kuperwasser

    The transcription factor Snai2, encoded by the SNAI2 gene, is an evolutionarily conserved C2H2 zinc finger protein that orchestrates biological processes critical to tissue development and tumorigenesis. Initially characterized as a prototypical epithelial-to-mesenchymal transition (EMT) transcription factor, Snai2 has been shown more recently to participate in a wider variety of biological processes, including tumor metastasis, stem and/or progenitor cell biology, cellular differentiation, vascular remodeling and DNA damage repair. The main role of Snai2 in controlling such processes involves facilitating the epigenetic regulation of transcriptional programs, and, as such, its dysregulation manifests in developmental defects, disruption of tissue homeostasis, and other disease conditions. Here, we discuss our current understanding of the molecular mechanisms regulating Snai2 expression, abundance and activity. In addition, we outline how these mechanisms contribute to disease phenotypes or how they may impact rational therapeutic targeting of Snai2 dysregulation in human disease.

    更新日期:2019-11-01
  • ER-associated degradation in health and disease - from substrate to organism.
    J. Cell Sci. (IF 4.401) Pub Date : null
    Asmita Bhattacharya,Ling Qi

    The recent literature has revolutionized our view on the vital importance of endoplasmic reticulum (ER)-associated degradation (ERAD) in health and disease. Suppressor/enhancer of Lin-12-like (Sel1L)-HMG-coA reductase degradation protein 1 (Hrd1)-mediated ERAD has emerged as a crucial determinant of normal physiology and as a sentinel against disease pathogenesis in the body, in a largely substrate- and cell type-specific manner. In this Review, we highlight three features of ERAD, constitutive versus inducible ERAD, quality versus quantity control of ERAD and ERAD-mediated regulation of nuclear gene transcription, through which ERAD exerts a profound impact on a number of physiological processes.

    更新日期:2019-11-01
  • Amyloid assembly and disassembly.
    J. Cell Sci. (IF 4.401) Pub Date : 2018-04-15
    Edward Chuang,Acacia M Hori,Christina D Hesketh,James Shorter

    Amyloid fibrils are protein homopolymers that adopt diverse cross-β conformations. Some amyloid fibrils are associated with the pathogenesis of devastating neurodegenerative disorders, including Alzheimer's disease and Parkinson's disease. Conversely, functional amyloids play beneficial roles in melanosome biogenesis, long-term memory formation and release of peptide hormones. Here, we showcase advances in our understanding of amyloid assembly and structure, and how distinct amyloid strains formed by the same protein can cause distinct neurodegenerative diseases. We discuss how mutant steric zippers promote deleterious amyloidogenesis and aberrant liquid-to-gel phase transitions. We also highlight effective strategies to combat amyloidogenesis and related toxicity, including: (1) small-molecule drugs (e.g. tafamidis) to inhibit amyloid formation or (2) stimulate amyloid degradation by the proteasome and autophagy, and (3) protein disaggregases that disassemble toxic amyloid and soluble oligomers. We anticipate that these advances will inspire therapeutics for several fatal neurodegenerative diseases.

    更新日期:2019-11-01
  • The filamin-B-refilin axis - spatiotemporal regulators of the actin-cytoskeleton in development and disease.
    J. Cell Sci. (IF 4.401) Pub Date : 2018-04-15
    Jacques Baudier,Zandra A Jenkins,Stephen P Robertson

    During development, cycles of spatiotemporal remodeling of higher-order networks of actin filaments contribute to control cell fate specification and differentiation. Programs for controlling these dynamics are hard-wired into actin-regulatory proteins. The filamin family of actin-binding proteins exert crucial mechanotransduction and signaling functions in tissue morphogenesis. Filamin-B (FLNB) is a key player in chondrocyte progenitor differentiation for endochondral ossification. Biallelic loss-of-function mutations or gain-of-function mutations in FLNB cause two groups of skeletal disorders that can be attributed to either the loss of repressive function on TGF-β signaling or a disruption in mechanosensory properties, respectively. In this Review, we highlight a unique family of vertebrate-specific short-lived filamin-binding proteins, the refilins (refilin-A and refilin-B), that modulate filamin-dependent actin crosslinking properties. Refilins are downstream TGF-β effectors in epithelial cells. Double knockout of both refilin-A and refilin-B in mice results in precocious ossification of some axial skeletal elements, leading to malformations that are similar to those seen in FLNB-deficient mice. Based on these findings, we present a model summarizing the role of refilins in regulating the mechanosensory functions of FLNB during skeletal development. We also discuss the possible contribution of refilins to FLNB-related skeletal pathologies that are associated with gain-of-function mutations.

    更新日期:2019-11-01
  • Axonal mRNA transport and translation at a glance.
    J. Cell Sci. (IF 4.401) Pub Date : 2018-04-15
    Pabitra K Sahoo,Deanna S Smith,Nora Perrone-Bizzozero,Jeffery L Twiss

    Localization and translation of mRNAs within different subcellular domains provides an important mechanism to spatially and temporally introduce new proteins in polarized cells. Neurons make use of this localized protein synthesis during initial growth, regeneration and functional maintenance of their axons. Although the first evidence for protein synthesis in axons dates back to 1960s, improved methodologies, including the ability to isolate axons to purity, highly sensitive RNA detection methods and imaging approaches, have shed new light on the complexity of the transcriptome of the axon and how it is regulated. Moreover, these efforts are now uncovering new roles for locally synthesized proteins in neurological diseases and injury responses. In this Cell Science at a Glance article and the accompanying poster, we provide an overview of how axonal mRNA transport and translation are regulated, and discuss their emerging links to neurological disorders and neural repair.

    更新日期:2019-11-01
  • Characterization of the novel mitochondrial genome replication factor MiRF172 in Trypanosoma brucei.
    J. Cell Sci. (IF 4.401) Pub Date : 2018-04-08
    Simona Amodeo,Martin Jakob,Torsten Ochsenreiter

    The unicellular parasite Trypanosoma brucei harbors one mitochondrial organelle with a singular genome called the kinetoplast DNA (kDNA). The kDNA consists of a network of concatenated minicircles and a few maxicircles that form the kDNA disc. More than 30 proteins involved in kDNA replication have been described. However, several mechanistic questions are only poorly understood. Here, we describe and characterize minicircle replication factor 172 (MiRF172), a novel mitochondrial genome replication factor that is essential for cell growth and kDNA maintenance. By performing super-resolution microscopy, we show that MiRF172 is localized to the kDNA disc, facing the region between the genome and the mitochondrial membranes. We demonstrate that depletion of MiRF172 leads to a loss of minicircles and maxicircles. Detailed analysis suggests that MiRF172 is involved in the reattachment of replicated minicircles to the kDNA disc. Furthermore, we provide evidence that the localization of the replication factor MiRF172 not only depends on the kDNA itself, but also on the mitochondrial genome segregation machinery, suggesting an interaction between the two essential entities.This article has an associated First Person interview with the first author of the paper.

    更新日期:2019-11-01
  • Loss of PTEN promotes formation of signaling-capable clathrin-coated pits.
    J. Cell Sci. (IF 4.401) Pub Date : 2018-03-29
    Luciana K Rosselli-Murai,Joel A Yates,Sei Yoshida,Julia Bourg,Kenneth K Y Ho,Megan White,Julia Prisby,Xinyu Tan,Megan Altemus,Liwei Bao,Zhi-Fen Wu,Sarah L Veatch,Joel A Swanson,Sofia D Merajver,Allen P Liu

    Defective endocytosis and vesicular trafficking of signaling receptors has recently emerged as a multifaceted hallmark of malignant cells. Clathrin-coated pits (CCPs) display highly heterogeneous dynamics on the plasma membrane where they can take from 20 s to over 1 min to form cytosolic coated vesicles. Despite the large number of cargo molecules that traffic through CCPs, it is not well understood whether signaling receptors activated in cancer, such as epidermal growth factor receptor (EGFR), are regulated through a specific subset of CCPs. The signaling lipid phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3], which is dephosphorylated by phosphatase and tensin homolog (PTEN), is a potent tumorigenic signaling lipid. By using total internal reflection fluorescence microscopy and automated tracking and detection of CCPs, we found that EGF-bound EGFR and PTEN are enriched in a distinct subset of short-lived CCPs that correspond with clathrin-dependent EGF-induced signaling. We demonstrated that PTEN plays a role in the regulation of CCP dynamics. Furthermore, increased PI(3,4,5)P3 resulted in higher proportion of short-lived CCPs, an effect that recapitulates PTEN deletion. Altogether, our findings provide evidence for the existence of short-lived 'signaling-capable' CCPs.

    更新日期:2019-11-01
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