Loss of MYO5B causes mitotic spindle orientation defects and cytokinesis delay

In order to investigate the effects of loss of MYO5B on mitotic spindle orientation, we generated MYO5B knockout human intestinal epithelial Caco2 cells using CRISPR-Cas9 technology. Knockout of the MYO5B gene was verified by the absence of the encoded myosin Vb protein on Western blot (see S1A Fig) and supported by sequencing of the MYO5B gene, which revealed the introduction of a premature termination codon in exon 3 (see S1B Fig). MYO5B knockout cells (hereafter referred to as Caco2^MYO5B−/−) and MYO5B wild-type (WT) cells (hereafter referred to as Caco2^WT) were cultured on coverslips and transfected with mCherry-tagged Histone2B to visualize chromosomes and GFP-tagged β-tubulin or, alternatively, labeled with antibodies against these proteins in order to visualize the mitotic spindle apparatus by laser scanning confocal fluorescence microscopy. The orientation of the mitotic spindle relative to the substratum (x-z direction) in cell monolayers was determined in 3D-reconstructed confocal images(x-y direction) of fixed cells by measuring the angle (β) between a line drawn through the spindle poles and a line parallel to the substratum (Fig 1A). The results show that metaphase Caco2^WT cells showed an average β-angle of 6° relative to the substratum, and a β-angle beyond 20° was rarely observed (Fig 1B). In contrast, metaphase Caco2^MYO5B−/− cells showed an average β-angle of 12° and a significantly higher percentage of cells displayed β-angles beyond 20° and up to 40° (Fig 1A, Fig 1B). This increase in the frequency of tilted mitotic spindles in metaphase Caco2^MYO5B−/− cells persisted, albeit to a lesser extent, through subsequent anaphase (Fig 1C, Fig 1D). Aberrant spindle tilting can result in the delamination of epithelial cells from the monolayer and loss of epithelial polarity of delaminated cells [15–17,19,20]. In accordance, in comparison to Caco2^WT cells, Caco2^MYO5B−/− cell cultures showed an increased percentage of cells that were placed on top of the monolayer (Fig 1E, Fig 1F) and which had lost basolateral surface polarity as evidenced by the nonpolarized distribution of the basolateral resident Na⁺/K⁺-ATPase (Fig 1G). Similar results were obtained when the cells were plated as a monolayer on semipermeable Transwell filter supports (see S2 Fig).

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Fig 1. Loss of MYO5B causes mitotic spindle orientation defects.

(A) Caco2^WT and Caco2^MYO5B−/−cells in metaphase were fixed and stained as indicated. The β-angle represents the angle between the spindle axis and the substratum in the confocal x-z dimension. (B) The β-angle was quantified in metaphase. (Left side graph) Each dot indicates one cell’s β-angle. (Right side graph) The statistical analysis of the mean for each experiment. n ≥ 15 mitotic cells/experiment were analyzed for N = 3 independent experiments. Values for each data point can be found in S1 Data. (C) Caco2^WT and Caco2^MYO5B−/− cells in anatelophase were fixed and stained as indicated. (D) The quantification of β-angle in anatelophase. n ≥ 9 mitotic cells/experiment were analyzed for N = 3 independent experiments. Values for each data point can be found in S1 Data. (E–F) The presence of cells not contacting the substratum was indicated by asterisks in nuclei (E). The percentage of nonsubstrate-contacting cells was quantified. Values for each data point can be found in S1 Data (F). (G) Na⁺/K⁺ ATPase staining shows basolateral localization in substrate contacting cells but not in multilayered cells. N = 3 independent experiments. t test,*p < 0.05, **p < 0.01, ***p < 0.001. Error bars indicate ± SEM (dot graph) or + SD (bar graph). Scale bars: 5 μm.

https://doi.org/10.1371/journal.pbio.3000531.g001

In addition to the tilted spindle phenotype, Caco2^MYO5B−/− cells showed a higher cytokinesis index when compared with Caco2^WT cells, as evidenced by the presence of a β-tubulin-positive cytokinesis bridge (Fig 2A). Live cell imaging of mCherry-H2B/GFP-β-tubulin-expressing cells revealed that cytokinesis was delayed in Caco2^MYO5B−/− cells (Fig 2B, see S1 Video, S2 Video). Thus, measuring the time that passed from the first appearance until the disappearance of the GFP-β-tubulin-positive cytoplasmic bridge showed an on average 2-fold increase (from 40 to 90 min in Caco2^WT and Caco2^MYO5B−/− cells, respectively) in cytokinesis duration (Fig 2C).

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Fig 2. Loss of MYO5B causes cytokinesis delay.

(A) Caco2^WT and Caco2^MYO5B−/−cells were fixed and stained as indicated. The percentage of cytokinesis with both daughter cells was quantified. n > 1,000 cells/experiment were analyzed for N = 3 independent experiments. Values for each data point can be found in S2 Data. (B) Live cell imaging shows x-y-t time-lapse mitosis images on Caco2^WT and Caco2^MYO5B−/− cells expressing β-tubulin-GFP and histone2B-mCherry. (C) The time of cytokinesis duration was quantified. (Left side graph) Each dot indicates 1 mitotic cell’s cytokinesis time. (Right side graph) The statistical analysis of the mean for each experiment. n ≥ 20 cells/experiment were analyzed for N = 3 independent experiments. Values for each data point can be found in S2 Data. t test, *p < 0.05, **p < 0.01, ****p < 0.0001. Error bars indicate ± SEM (dot graph) or + SD (bar graph). Scale bars: 5 μm.

https://doi.org/10.1371/journal.pbio.3000531.g002

In conclusion, mitotic spindle orientation defects, and cytokinesis delays were observed in Caco2 cells lacking MYO5B expression.

Concurrence of mitotic spindle orientation defects and the presence of large vacuoles in MYO5B-depleted cells

Upon closer microscopical inspection of cells with tilted mitotic spindles, we noticed that virtually all Caco2^MYO5B−/− cells with tilted spindles contained one or more large (i.e., Ø >1 μm) vacuoles (Fig 3A, arrows and dotted white circles [note that this is the same image as in Fig 1A]). Subsequent comparison of the β-angles in Caco2^MYO5B−/− cells with a vacuole or without a vacuole revealed that the increase in spindle tilting during metaphase and anaphase was restricted to those Caco2^MYO5B−/− cells that displayed a vacuole (Fig 3B, Fig 3C). The absence of a spindle orientation defect in the small intestine of Myo5b knockout mice (see S3 Fig), which do form vacuoles but in villus enterocytes and not in the proliferative crypt cells (see below), supports that the mere loss of Myo5b expression (i.e., without a vacuole present) did not cause the spindle misorientation phenotype.

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Fig 3. Mitotic spindle orientation defects correlate with the presence of large vacuoles in MYO5B-depleted cells.

(A) Caco2^MYO5B−/− cells were fixed and stained as indicated. Arrows in bright field and dotted circles in fluorescent field indicate large vacuoles in Caco2^MYO5B−/− cells during metaphase and ana-telophase. (B) The β-angle in Caco2^MYO5B−/− cells with vacuole and without vacuole during metaphase was quantified. (Left side graph) Each dot indicates one mitotic cell’s metaphase β-angle. (Right side graph) The statistical analysis of the mean for each experiment. n ≥ 8 mitotic cells/experiment were analyzed for N = 3 independent experiments. Values for each data point can be found in S3 Data. (C) The quantification of β-angle in Caco2^MYO5B−/− cells with vacuole and without vacuole during anatelophase. n ≥ 6 cells/experiment were analyzed for N = 3 independent experiments. Values for each data point can be found in S3 Data. t test, **p < 0.01, ****p < 0.0001. Error bars indicate ± SEM (dot graph) or + SD (bar graph). Scale bars: 5 μm.

https://doi.org/10.1371/journal.pbio.3000531.g003

We then subjected Caco2^MYO5B−/− cells to live cell fluorescence imaging in the x-y plane to investigate the effect of the vacuole on spindle orientation dynamics in real time. In Caco2^MYO5B−/− cells without a vacuole, spindle rotation dynamics were minimal during metaphase (Fig 4A, see S3 Video). Thus, the average angle (α) between a line drawn through the spindle axis at the onset of metaphase and the end of metaphase (i.e., the onset of anaphase) was approximately 10°. By contrast, in Caco2^MYO5B−/− cells that contained a vacuole, the average α-angle was approximately 25° (Fig 4B). Reconstructed movies of images captured serially in time showed a dynamic reorientation of the mitotic spindle relative to the position of the vacuole (Fig 4A; see S4 Video). Quantitative analysis of still images taken from Caco2^MYO5B−/− cells that displayed increased spindle rotation dynamics (i.e., an α-angle of more than 25°; c.f., Fig 4B) revealed an increase in the distance (d) measured between the vacuole rim and the spindle pole (Fig 4C) during the course of metaphase (Fig 4D and 4E, see S5 Video), supporting that the orientation of the mitotic spindle changed relative to the position of the vacuole. Thus, the presence of the vacuole influenced mitotic spindle orientation.

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Fig 4. Mitotic spindle orientation defects correlated to the presence of large vacuoles in MYO5B-depleted cells.

(A) Live cell imaging shows mitosis images on Caco2^MYO5B−/−cells expressing β-tubulin-GFP and histone2B-mCherry. The α angle represents the angle between a line drawn through the spindle axis at the onset of metaphase (dotted line) and the end of metaphase (solid line). Dotted circles indicate large vacuoles which were confirmed by bright field. (B) The α angle in Caco2^MYO5B−/−cells with vacuole and without vacuole during metaphase was quantified. Each dot indicating 1 mitotic cell’s spindle rotation α angle (left). The statistical analysis of the mean for each experiment (right). n > 15 cells/experiment were analyzed for N = 3 independent experiments. Values for each data point can be found in S4 Data. (C) The position of mitotic spindle relative to vacuole during onset and end of metaphase in Caco2^MYO5B−/− cells was quantified by measuring the distance from spindle pole to vacuole (d, red line). Arrows in the bright field and dotted circles in the fluorescent field indicate large vacuoles. (D) The comparison of the distance d in the onset and end of metaphase in Caco2^MYO5B−/− cells. Values for each data point can be found in S4 Data. (E) The quantification of the distance d in vacuolated Caco2^MYO5B−/− cells. (Left side graph) Each dot indicates one cell’s distance d between the onset and end of metaphase. (Right side graph) The statistical analysis of the mean for each experiment. n ≥ 5 cells/experiment were analyzed for N = 3 independent experiments. Values for each data point can be found in S4 Data. t test, **p < 0.01, ****p < 0.0001. Error bars indicate ± SEM (dot graph) or + SD (bar graph). Scale bars: 5 μm.

https://doi.org/10.1371/journal.pbio.3000531.g004

In addition to the mitotic spindle tilting, the presence of a large vacuole was also correlated with an increased frequency of 2 other mitotic complications: (1) a modest increase (from approximately 3% to approximately 8% of WT and knockout cells, respectively) in metaphase arrest frequency (see S4A–S4C Fig and S6 Video; note that, in cells that were not arrested in metaphase during the time span of the experiment, the presence of the vacuole did not affect the average duration of metaphase [S2A and S2B Fig]) and (2) an increase (from approximately 5% to approximately 12% of WT and knockout cells, respectively) in chromosome segregation difficulties (i.e., chromosome lagging, chromosome bridging, or micronuclei; see S5 Fig). Finally, vacuoles were also observed in Caco2^MYO5B−/− cells during cytokinesis, but no statistically significant difference in cytokinesis index between Caco2^MYO5B−/− cells with or without a vacuole was observed (see S4D and S4E Fig).

Thus, the mitotic spindle tilting defect and the increased incidence of metaphase arrest and chromosome segregation difficulties—but not the delay in cytokinesis—as observed in MYO5B-depleted cells was strongly correlated to the presence of large vacuoles.

Loss of MYO5B expression causes the formation of giant late endosomes

We then focused on the identity of the vacuoles. We found that Caco2^MYO5B−/− cells, as such, displayed significantly more large vacuoles in the cytoplasm when compared with Caco2^WT cells (Fig 5A), and this was observed in multiple MYO5B knockout clones (Fig 5B). Large vacuoles were observed in approximately 30% of all fixed cells (Fig 5A). Live cell imaging, however, revealed that <20% of the Caco2^WT but >80% of the Caco2^MYO5B−/− cells formed a vacuole (Ø > 1 μm) for at least 30 min during the 24 h time course of the experiments (Fig 5C). Electron microscopy analysis confirmed the presence of large electron-lucent vacuoles in Caco2^MYO5B−/− cells (Fig 5D), and the presence of intraluminal material suggested resemblance to degradative compartments. Immunolabeling with antibodies against various organelle markers showed that the vacuoles were specifically positive for late endosome markers (i.e., late endosome–associated membrane protein [LAMP]1 and the small GTPase rab7) (Fig 5E and 5F; see S6 Fig and S7 Fig). No lysotracker or autophagosome markers, i.e., microtubule-associated protein 1 light chain (LC)-3B (Fig 5E and 5F) and sequestosome-1 (p62; see S6 Fig), were observed at the vacuoles, indicating that the vacuoles represented nonacidic and nonautophagic giant late endosomes. We also observed the presence of vacuolated LAMP1-positive late endosomes in villus enterocytes (but not in crypts) of the small intestine of Myo5b knockout mice and MVID patients carrying bi-allelic MYO5B mutations (see S8A–S8C Fig). Importantly, upon the re-introduction of myc-tagged full-length human myosin Vb in Caco2^MYO5B−/− cells, the vacuoles no longer formed (Fig 5G), demonstrating that the formation of the large vacuoles was causally related to the loss of MYO5B expression.

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Fig 5. Loss of MYO5B expression causes the formation of giant late endosomes in Caco2^MYO5B−/− cells.

(A) Arrows indicate large vacuoles in Caco2^MYO5B−/− cells. The percentage of cells with vacuole (diameter Ø > 1 μm) was quantified in fixed Caco2^WT and Caco2^MYO5B−/− cells. (B) Multiple Caco2^MYO5B−/− clones showed a higher percentage of cells with vacuoles compared with heterozygous or control clones. (C) The percentage of cells formed a vacuole (Ø > 1 μm) for at least 30 min during the 24 h was quantified in live cell imaging experiments. (D) Electron microscopy images of Caco2^MYO5B−/− cells revealed the large electron-lucent vacuoles (arrows) were with single membrane (black thick arrow) and presence of intraluminal material (white thick arrow). (E–F) Vacuoles (arrows) in Caco2^MYO5B−/− cells were positive for Rab7 and LAMP1 but virtually negative for lysotracker, LC-3B, and other various organelle markers. For using lysotracker, Caco2^MYO5B−/− cells were incubated in medium with lysotracker for 1 h before fixation. (G) Western blot showing myosin Vb expression after the re-expression of a CRISPR-Cas9–resistant human MYO5B cDNA in Caco2^MYO5B−/− cells (Caco2^MYO5B−/− rescue cells), and the ameliorating effect on the number of vacuoles. (A, C, F and G) N = 3 independent experiments. t test, ***p < 0.001. Error bars indicate + SD (bar graph). (A and E) Scale bars: 10 μm. Values for each data point in panels A, B, C, F, and G can be found in S5 Data. EEA, early endosomal antigen-1; KD, knockdown; LC, light chain; ns, not significant; WT, wild type.

https://doi.org/10.1371/journal.pbio.3000531.g005

We also generated MYO5B knockout human embryonic kidney (HEK)293 cells (HEK293^MYO5B−/−; see S9A Fig). When compared to WT HEK293 cells, HEK293^MYO5B−/− developed large vacuoles (S9B Fig), displayed therewith correlated mitotic spindle tilting (S9C–S9G Fig) and cell delamination (S10A and S10B Fig). Further, HEK293^MYO5B−/− displayed vacuole-unrelated delayed cytokinesis (S10C–S10E Fig). These results indicate that the observed effects were not specific for Caco2 cells.

We reasoned that understanding the mechanism that underlie the formation of giant late endosomes may provide tools to interfere with their formation and, hence, allow us to further test the relationship between the presence of the vacuoles and the mitotic spindle orientation defects. The distended appearance of the late endosomes suggested an ion imbalance and resembled the swollen late endosomes as seen in cells infected with Helicobacter pylori. H. pylori is a bacterium that inserts a vacuolating toxin with chloride channel activity into the host cell’s endosomal system and in this way causes late endosome ion imbalance and resultant late endosome swelling [21–23]. Indeed, inhibition of the vacuolar H⁺-adenosine triphosphatase (vATPase) proton pump activity with bafilomycin A1 (BafA1) or inhibition of chloride channel activity with either of 3 unrelated compounds (i.e., furosemide, 4,4'-Diisothiocyano-2,2'-stilbenedisulfonic acid [DIDS] and 5-nitro-2-(3-phenylpropyl-amino) benzoic acid [NPPB]) effectively and reversibly abolished the appearance of giant late endosomes in Caco2^MYO5B−/− cells (Fig 6A and 6B). Possibly, late endosome ion imbalance in MYO5B-depleted cells is the result of inhibited plasma membrane recycling of various ion transporters and their resultant accumulation in late endosomes. Indeed, loss of MYO5B expression is known to inhibit plasma membrane protein recycling and result in the appearance of plasma membrane proteins in late endosomes. Furthermore, in support of this, we observed a redistribution of a chloride channel, anoctamin (ANO)-6 [24,25], from the cell periphery to (giant) late endosomes upon loss of MYO5B expression (Fig 6C and 6D).

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Fig 6. vATPase and chloride channel activities mediate the late endosomal phenotype in Caco2^MYO5B−/− cells.

(A–B) The numbers of vacuoles in Caco2^MYO5B–/–cells were reduced after treatments with vATPase inhibitor BafA1 and chloride channel inhibitors NPPB, DIDS, and furosemide for 24 h (A) and other appropriate times (B). Vacuoles reappeared after washout of the chemicals and culture in normal cell culture medium (B, arrow). Values for each data point can be found in S6 Data. (C–D) Caco2^MYO5B−/− cells revealed a shift in the abundance of the chloride channel ANO6 from the cell periphery to LAMP1-positive compartments (C, thick arrows) and vacuoles (D, thin arrows) compared with Caco2^WT cells. N = 3 independent experiments. Scale bars: 10 μm. ANO, anoctamin; Baf, bafilomycin; DIDS, 4,4'-Diisothiocyano-2,2'-stilbenedisulfonic acid; LAMP, late endosome–associated membrane protein; NPPB, 5-nitro-2-(3-phenylpropyl-amino) benzoic acid; vATPase, vacuolar H⁺-adenosine triphosphatase; WT, wild type.

https://doi.org/10.1371/journal.pbio.3000531.g006

Notably, treatment of Caco2^MYO5B−/− cells with BafA1 not only rescued the giant late endosome phenotype but also normalized the tilted spindle orientation to control values (Fig 7A–7C). Treatment of WT cells with BafA1 did not affect spindle orientation (Fig 7D and 7E). Further treatment of Caco2^MYO5B−/− cells with BafA1 abolished cell delamination (Fig 7F and 7G). These results support the relationship between the presence of the vacuoles and the mitotic spindle orientation defects.

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Fig 7. vATPase inhibitor Baf A1 rescues the spindle orientation defects in Caco2^MYO5B−/− cells.

(A) Caco2^WT and Caco2^MYO5B−/− cells were treated with Baf A1 for 24 h before fixation and stained as indicated. The β-angle represents the angle between the spindle axis and the substratum in the confocal x-z dimension. (B–E) The β-angle in metaphase and anatelophase was quantified in Caco2^WT and Caco2^MYO5B−/− cells after treated with Baf A1. The dot graph: each dot indicates one cell’s β-angle. The histogram graph: the statistical analysis of the mean for each experiment. n ≥ 9 cells/experiment were analyzed for N = 3 independent experiments. (B) Caco2^MYO5B−/− in metaphase, (C) Caco2^MYO5B−/− in anatelophase, (D) Caco2^WT in metaphase, (E) Caco2^WT in anatelophase, (F, G) The percentage of nonsubstrate-contacting cells was quantified in Caco2^WT and Caco2^MYO5B−/− cells after treated with Baf A1. N = 3 independent experiments. t test, *p < 0.05,**p < 0.01. Error bars indicate ± SEM (dot graph) or + SD (bar graph). Scale bars: 5 μm. Values for each data point in panels B, C, D, E and G can be found in S7 Data. baf,; ns, not significant; WT, wild type.

https://doi.org/10.1371/journal.pbio.3000531.g007

Rab7 availability controls the formation of giant late endosomes and mitotic spindle orientation defects in MYO5B-depleted cells

Rab7 is a key regulator of late endosome dynamics [26] and has been reported to correct trafficking defects in lysosomal storage disease when overexpressed [27]. Our data demonstrate that the overexpression of an enhanced green fluorescent protein (eGFP)-tagged WT(rab7-WT) or eGFP-tagged dominant-negative (DN) rab7 mutant (rab7-T22N) in Caco2^MYO5B−/− cells inhibited or exaggerated the formation of giant late endosomes, respectively (Fig 8A and 8B).

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Fig 8. Rab7 controls the formation of giant endosomes.

(A, B) Quantification of the vacuoles (A, arrows) percentage of Caco2^MYO5B–/–cells expressing GFP-rab7-WT or GFP-rab7-T22N. N = 3 independent experiments. Values for each data point can be found in S8 Data. Student t test, *p < 0.05, ***p < 0.001. Error bars indicate + SD. Scale bars: 10 μm. GFP, green fluorescent protein; WT, wild type.

https://doi.org/10.1371/journal.pbio.3000531.g008

We took advantage of the modifying effects of rab7 to further address the causality between the presence of giant late endosomes and mitotic spindle orientation defects upon the loss of MYO5B expression. We found that the overexpression of rab7-WT, but not of the inactive rab7-T22N mutant, in Caco2^MYO5B−/− cells not only effectively prevented the formation of giant late endosomes but also normalized the mitotic spindle tilting range relative to the substratum (β-angle) in metaphase and anaphase cells (Fig 9A–9C). Furthermore, the overexpression of rab7-WT, but not of rab7-T22N, prevented the increase in Caco2^MYO5B−/− cell delamination (Fig 9D and 9E). The expression of rab7-WT or rab7-T22N in Caco2^MYO5B−/− cells did not affect the observed delay in cytokinesis (see S11 Fig), which is in agreement with the nonsignificant correlation between the presence of giant late endosomes and cytokinesis duration. Together these results indicate a causal relationship between the presence of giant late endosomes, spindle orientation defects, and epithelial cell delamination, but not cytokinesis delay, in cells that have lost MYO5B expression.

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Fig 9. Rab7 controls the formation of giant endosomes and mitotic spindle orientation defects in Caco2^MYO5B−/− cells.

(A) Caco2^MYO5B−/− cells expressing GFP-rab7-WT or GFP-rab7-T22N in metaphase and anatelophase were fixed and stained as indicated. Arrows in bright field and dotted circles in fluorescent field indicate large vacuoles. (B) The β-angle was quantified in Caco2^MYO5B−/− cells expressing GFP-rab7-WT or GFP-rab7-T22N during metaphase and anatelophase. (Upper graph) Each dot indicates one cell’s β-angle. (Bottom graph) The statistical analysis of the mean for each experiment. n ≥ 6 cells/experiment were analyzed for N = 3 independent experiments. (C) The quantification of β-angle in Caco2^MYO5B−/− cells expressing GFP-rab7-T22N with and without vacuoles. (Upper graph) Each dot indicates one cell’s β-angle. (Bottom graph) The statistical analysis of the mean for each experiment. n ≥ 6 cells/experiment were analyzed for N = 3 independent experiments. (D–E) The percentage of cells not contacting the substratum (D, asterisks) was quantified in Caco2^MYO5B−/− cells expressing GFP-rab7-WT or GFP-rab7-T22N. N = 3 independent experiments. t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars indicate ± SEM (dot graph) or + SD (bar graph). Scale bars: 5 μm. Values for each data point in panels B, C, and E can be found in S9 Data. GFP, green fluorescent protein; ns, not significant; WT, wild type.

https://doi.org/10.1371/journal.pbio.3000531.g009

Wortmannin-induced giant endosomes correlate with mitotic spindle orientation defects

We reasoned that if giant endosomes, rather than the mere loss of MYO5B, was responsible for the mitotic spindle orientation phenotype, the induction of giant endosomes via a different mechanism should also result in the spindle orientation phenotype. To address this issue, we treated WT Caco2 cells with wortmannin (Wm), a fungal metabolite and potent inhibitor of phosphatidylinositol-3 kinases that was previously reported to induce endosomal vacuoles in different cell types [28–30]. Treatment of WT Caco2 cells with Wm induced the formation of large endosomal vacuoles with comparable sizes as seen in MYO5B-depleted cells (Fig 10A). Antibody labeling showed that these vacuoles were positive for markers of both early (EEA1) and late (rab7, LAMP1) endosomes (Fig 10B). Wm-treated WT Caco2 cells that formed large vacuoles displayed a mitotic spindle tilting phenotype similar to that seen in vacuole-containing MYO5B-depleted cells (Fig 10C–10E). These data thus support a physical occlusion model in which the presence of giant endosomes, rather than other myosin Vb-mediated processes, caused tilting of the mitotic spindle apparatus.

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Fig 10. PI3K inhibitor Wm induces large vacuoles and mitotic spindle orientation defects in Caco2^WT cells.

(A) The numbers of Caco2^WT cells with vacuoles (diameter Ø > 1 μm) are increased after treated with Wm for 1 h. (B) The vacuoles (arrows) induced by Wm are positive for LAMP1, Rab7 and EEA1. (C) Caco2^WT cells treated with Wm were fixed and stained as indicated. The dotted circles in the images indicate large vacuoles during metaphase and anatelophase. (D, E) The β-angle in metaphase (D) and anatelophase (E) was quantified in Caco2^WT cells after being treated with Wm. The dot graph: each dot indicates one cell’s β-angle. The histogram graph: the statistical analysis of the mean for each experiment. More than 9 cells/experiment were analyzed for N = 3 independent experiments. t test, **p < 0.01, ****p < 0.0001. Error bars indicate ± SEM (dot graph) or + SD (bar graph). Scale bars: 5 μm. Values for each data point in panels A, D, and E can be found in S10 Data. EEA, early endosomal atigen-1; LAMP, late endosome–associated membrane protein; Wm, Wm; WT, wild type.

https://doi.org/10.1371/journal.pbio.3000531.g010

Ethics statement

All animal experiments comply with the Guidelines of the European Union Council (2010/63/UE) and of the Spanish Government (RD 53/2013) and were approved by the Ethical committee of Animal Experimentation at Vall d’Hebron Institute of Research (80/12 CEEA).

Cells and tissues

Human Caco2 cells (HTB-37) and HEK293 cells (CRL-1573) (ATCC, Gaithersburg, MD, USA)were grown in DMEM (Thermo Scientific Fisher, Waltham, MA, USA), supplemented with 10% heat-inactivated fetal calf serum (Invitrogen) and antibiotics (penicillin, streptomycin) (Thermo Scientific Fisher, Waltham, MA, USA), and maintained at 37°C in a humidified atmosphere with 5% CO₂. For experiments, cells were typically cultured in Lab-Tek chambers (Thermo Scientific, Waltham, MA, USA) or on Transwell semipermeable polycarbonate filter supports (Corning, New York, NY, USA3401). Tissues from MVID patients and age-matched control were described by Szperl and colleagues, Golachawska and colleagues, and Dhekne and colleagues [56–58].

Reagents and plasmids

Commercial antibodies used for immunofluorescence and Western blot are listed in S1 Table. The plenti-plasmids Rab7-WT-GFP and Rab7-GFP-T22N DN mutant were gifts from Peter van der Sluijs (University Medical Center Utrecht, the Netherlands). The plenti-plasmids β-Tubulin-GFP (64060) and Histone2B-mCherry (51007) are from Addgene (Watertown, MA, USA).

Virus production and transduction

Lentiviral particles were created using a second-generation system. Briefly, following the instruction of Fugene (Promega, Madison, WI, USA), the mixture of pLenti-plasmid, vesicular stomatitis virus-glycoprotein, delta-8.91, and Fugene was added in the 6-well plate, and 1 × 10⁶ HEK293T cells were seeded on top of the mixture. After overnight incubation, medium was changed with DMEM with 2.5% (v/v) serum and 1% antibiotics. After 48 h, viral particles were harvested and filtered through 0.45-μm filter (GE healthcare, Hatfield, UK). Cells were transduced by filtered virus supernatant with polybrene (8ug/ml) 6 h after seeding. The next day virus medium was changed with selection medium containing appropriate antibiotics.

Generation of cell lines

MYO5B knockout cells were generated using the CRISPR/Cas9 genome editing system. DNA Oligos were the following: sense: caccgGATATCTGGATTCCGTAAGA; antisense: aaacTCTTACGGAATCCAGATATCc.

Virus production and cell transduction procedures were as described above. Transduced cells were selected with puromycin (5 μg/ml, Sigma-Aldrich, St. Louis, MO, USA) 1 day after infection. After 3 days, infected cells were sparsely plated, and individual clones were picked. Finally, the presence of a premature stop codon was checked by sequencing (GATC Company), and myosin Vb expression levels were checked by western blot. Caco2 cells expressing Rab7-WT-GFP or Rab7-GFP-T22N, β-Tubulin-GFP, and Histone2B-mCherry were generated by virus transduction with plenti-plasmids as described above.

MYO5B rescue assay

CRISPR-cas9–resistant MYO5B cDNA was generated by site-directed mutagenesis (NEB, Hitchin, UK), introducing a silent C>G mutation in the pENTR1a-MYO5B corresponding to the 60th amino acid in the WT myosin Vb protein. The primers used to mutate MYO5B cDNA were: forward–ACCAGCTGCCGTTCTTACGGA-, reverse–TGCGTTGTACATCAATTGGG-. The myc-tagged CRISPR-cas9–resistant MYO5BcDNA was transferred to pLenti-plasmid with blasticidin-resistance. Caco2^MYO5B−/− cells were transduced with virus containing the CRISPR-cas9−resistant MYO5B cDNA. After blasticidin (6 ug/ml, Thermo Scientific, Waltham, MA, USA) selection, the expression of the myc-tag was checked by western blotting and immunofluorescence.

Myo5b intestinal knockout mice

Myo5btm1c conditional Myo5b knockout mice on a C57BL/6 background were generated by crossing Myo5btm1a(KOMP)Wtsi mice [59] with C57BL/6 Tg(CAG-Flpo)1Afst (Flp delete; EMMA ID EM:05149) [60] animals. To obtain intestinal-specific Myo5b^−/− mice, Myo5btm1c animals were crossed with a C57BL/6 Tg(Vil-cre/ERT2)23Syr mice (Vil-CreERT2; The Jackson Laboratory Stock No: 020282) [61], carrying a tamoxifen inducible Cre recombinase expressed under the control of the Villin promoter. Myo5b deletion was achieved by a single i.p. injection of 4 mg tamoxifen (Sigma-Aldrich, St. Louis, MO, USA), and animals were killed by cervical dislocation on day 5 after Cre induction. The orientation of the mitotic spindle in small intestinal cells was determined in hematoxylin and eosin stained sections of WT and iKO mice by measuring the angle formed by mitotic metaphase/anaphase cells and the epithelial surface. At least 70 mitosis were scored in 4 female 10-week-old mice per group.

Western blotting

Cells were harvested with lysis buffer (100 mM NaCl, 20 mM Tris-HCl [pH 7.6]), Triton X-100 and protease inhibitors (Roche, Almere, the Netherlands). Protein concentration was determined using the BCA protein assay (Sigma-Aldrich, St. Louis, MO, USA). Protein extracts (20 μg) were resolved on an SDS/4-15% polyacrylamide gel, transferred to nitrocellulose membranes, and blocked with Odyssey Blocking Buffer (LI-COR, Lincoln, NE, USA) at room temperature for 1 h. The membrane was incubated with primary antibodies (see S1 Table) at 4°C for 16 h, washed 3 times with PBS with 0.1% (v/v) Tween, and incubated with the secondary antibody in OBB/PBS buffer at room temperature for 1 h. Following washes with PBS with 0.1% (v/v) Tween, antibody signal was detected using Odyssey Imaging System (LI-COR, Lincoln, NE, USA).

Immunofluorescence and confocal microscopy

For immunofluorescence microscopy, cells were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.2% (v/v) Triton X-100 at room temperature for 10 min. Cells were washed with PBS at each step. Cells were incubated in PBS with 1% (w/v) BSA at 37°C for 1 h, followed by overnight incubation with primary antibodies (see S1 Table) in PBS with 1% (w/v) BSA at 4°C. After washes with PBS, cells were incubated with secondary antibodies in PBS with 1% BSA (w/v) at room temperature for 1 h. Following washes with PBS, cells were mounted in Dako mounting medium and imaged using a fluorescence microscope (Leica, Frankfurt, Germany). In mitotic tilted experiments, cells were cultured in an 8-chambers plate (Lab-Tek II, 155409) and incubated with DMEM medium for 5 days. Cells were stained as above and imaged by TCS SP8 confocal microscope (Leica DMI 6000) (Leica, Frankfurt, Germany) with x-y-z stage.

Electron microscopy

Tissues were fixed in 2% glutaraldehyde in 0.1 M sodiumcacodylate buffer at 4°C for 24 h. Cells grown in 6-well plates were fixed by adding dropwise an equal volume fixative (2% [v/v] glutaraldehyde, 2% [v/v] paraformaldehyde in 0.1 M sodiumcacodylate buffer). After 10 min, this mixture was replaced by pure fixative at room temperature for 30 min. After postfixation in 1% osmium tetroxide/1.5% potassium ferrocyanide (v/v, 4°C, and 30 min), tissues and cells were dehydrated using ethanol and embedded in EPON epoxy resin. A total of 60 nm sections were cut and contrasted using 2% (v/v) uranyl acetate in water followed by Reynolds lead citrate. Images were taken with a Zeiss Supra 55 in STEM-mode at 26KV using an external scan generator (Fibics, Canada) yielding mosaics of large area scans at 2.5 nm pixel resolutions.

Immunohistochemistry

Immunohistochemistry was performed on 4 μm-thick tissue sections. The sections were deparaffinized, rehydrated in xylene and graded ethanol solutions. Sodium citrate buffer (10 mM sodium citrate, 0.05% (v/v) Tween-20, [pH 6.0]) was used for antigen retrieval, and slides were incubated overnight with primary antibodies (see S1 Table), followed by incubation with secondary antibodies and detection with horseradish peroxidase and 3,3'-diaminobenzidine. Slides were counterstained with hematoxylin and covered with mounting medium (Dako, Santa Clara, CA, USA).

Treatment cells with different inhibitors

In experiments with vATPase and chloride channel inhibitors, cells were seeded on the coverslips in a 24-well plate and incubated with DMEM medium for 72 h. The vATPase inhibitor Baf A1 (0.1 μM), chloride channel inhibitors NPPB (200 μM), DIDS (500 μM), and furosemide (2 mM; Sigma-Aldrich, St. Louis, MO, USA) were added into the medium. After incubated with chemicals for indicated times, cells were fixed with PFA, followed by mounting of the slides and microscopical examination. In experiments with the PI3K inhibitor Wm (Sigma-Aldrich, St. Louis, MO, USA), cells were cultured as above and incubated with Wm (500 nM) for 1 h before fixation. In mitotic tilting and delamination experiments, cells were cultured in an 8-chambers plate (Lab-Tek II, 155409) (Thermo Scientific, Waltham, MA, USA)and incubated with DMEM medium for 5 days. Cells were treated with Baf A1 (0.1 μM) for 24 h or Wm (500 nM) for 1 h before fixation. Then cells were immunolabeled as described above and imaged with a TCS SP8 confocal microscope (Leica DMI 6000) (Leica, Frakfurt, Germanywith x-y-z stage.

Live cell imaging

Cells expressing β-tubulin-GFP and histone2B-mCherry were seeded in an 8-chambers plate (Lab-Tek II, 155409) (Thermo Scientific, Waltham, MA, USA) and incubated with DMEM medium for 72 h. Then the live cells were observed 24 h with the laser scanning confocal microscope (GE Healthcare Bio-Sciences, Marlborough, MA, USA) with temperature and CO₂ control. Images were collected using a 40× oil objective with GFP-mCherry and DIC channels every 6 to 8 min. Images were processed by Imaris software (Oxford Instruments, Zurich, Switzerland; https://imaris.oxinst.com/downloads).

Quantification and statistical analysis

For live cell imaging and mitotic tilted experiments, images were performed with confocal microscope with x-y-z stage. In other immunofluorescence experiments, images were 2D object with x-y stage. All experiments were performed for at least 3 independent experiments. The measurements of the α-angle in horizontal rotation experiment were obtained with the free ImageJ tools (https://imagej.nih.gov/ij/download.html) in x-y confocal sections. The line of dividing direction was drawn vertically to the chromosome plane through the center in metaphase and drawn through the both the 2 daughter cells’ chromosome plane in anatelophase. The β-angle was obtained with the ImageJ tools x-z confocal sections. The indicated vacuole’s position was confirmed by bright field images. To estimate the percentage of cells not contacting the substratum, 5 random x-y fields of 4 × 10⁴ μm² were analyzed in the x-y-z dimensions for 3 independent experiments. For statistical estimation, we used GraphPad Prism version 7.0 software (Graphpad software, San Doego, CA, USA) (https://www.graphpad.com/scientific-software/prism). Graphs represent mean y ± SEM or + SD, as indicated, n represents the numbers of cells analyzed. Statistical significance was determined by unpaired 2-tailed t tests.