Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232.
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232.
Division of Infectious Diseases, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232.
Brush border microvilli enable functions that are critical for epithelial homeostasis, including solute uptake and host defense. However, mechanisms that regulate the assembly and morphology of these protrusions are poorly understood. The parallel actin bundles that support microvilli have their pointed-end rootlets anchored in a filamentous meshwork referred to as the “terminal web.” Although classic EM studies revealed complex ultrastructure, the composition and function of the terminal web remains unclear. Here we identify non-muscle myosin-2C (NM2C) as a component of the terminal web. NM2C is found in a dense, isotropic layer of puncta across the sub-apical domain, which transects the rootlets of microvillar actin bundles. Puncta are separated by ∼210 nm, the expected size of filaments formed by NM2C. In intestinal organoid cultures, the terminal web NM2C network is highly dynamic and exhibits continuous remodeling. Using pharmacological and genetic perturbations in cultured intestinal epithelial cells, we found that NM2C controls the length of growing microvilli by regulating actin turnover in a manner that requires a fully active motor domain. Our findings answer a decades old question on the function of terminal web myosin and hold broad implications for understanding apical morphogenesis in diverse epithelial systems.
Movie S1: Related to Figure 2. Live imaging of the apical surface of a 2D intestinal organoid monolayer derived from a NM2C-EGFP expressing mouse. Spinning disk confocal images were acquired every 60 seconds for 40 minutes; playback is 15 frames per second; field width is 53 μm.Download Original Video (19.7 MB)https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e20-09-0582/20201005/media/mc-e20-09-0582-s01.,1200,900,768,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518410abb28bf45a6fadfff54c0884ea58110075615c1d5919ea753725730a4097154a8f56c8491b2e70b0e87b5e9f35b4d61387d2130c8df350edbabb355e8ef985f87cef5b08e5fd566789b540170ceeb86822f94322bd65dfec29c52a90fc650e8eb2e1e77ce8d6d20b44e284747124ede2dca821a1319d74430a3da1c530cd51a207b3ec62c9518c42b07edef4f7800d77807e249b1346eb92b9cbee1f39692b78e7Movie S2: Related to Figure 3. Calyculin A shortens Ls174T-W4 cell microvilli. Spinning disk confocal imaging of an induced Ls174T-W4 cell expressing EGFP-UtrCH (magenta) and Halo-NM2C labeled with JF585 (green). Images were acquired every 15 seconds for 60 minutes; playback is 15 frames per second; field width is 23 μm.Download Original Video (19.9 MB)https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e20-09-0582/20201005/media/mc-e20-09-0582-s02.,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518515abb28bda92fbabe7b929571bd415190bf44d1790b7324fb90f184f91fab10e395327a1bef9725d0c99a358bbbe69c082a95f9d49957eb2932b2be9a6f328f5ec3ab3a0b9709f37f41cd72fbbabd2c3588c7488038615f2c4a4dbadb9df4030e588e612a3ac153bec21ed0aeb5bbc6f1579cafa57b97803098dfd04e4ae104a9a962bba87a8ad65c721868b54460a375387cf64bfMovie S3: Related to Figure 4. Blebbistatin elongates Ls174T-W4 cell microvilli. Spinning disk confocal imaging of an induced Ls174T-W4 cell expressing EGFP-UtrCH (magenta) and Halo-NM2C labeled with JF585 (green). Images were acquired every 30 seconds for 30 minutes; playback is 5 frames per second; field width is 29 μm.Download Original Video (7.0 MB)https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e20-09-0582/20201005/media/mc-e20-09-0582-s03.,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518515abb28bda92fbabe7b929571bd415190bf44d1790b7324fb90f184f91fab10e395327a1bef9725d0c99a358bbbe69c082a95f9d49957eb2932b2be9a6f328f5ec3ab3a0b9709f37f41cd72fbbabd2c3588c7488038615f2c4a4dbadb9df4030e588e612a3ac153bec21ed0aea5bbc6f1579cafa656e9b8335279ef2e5682956845ba6d9fd2ad30fa8df771ea9e47dd44f50996908Movie S4: Blebbistatin rescues microvilli shortened by Calyculin A. Spinning disk confocal imaging of an induced Ls174T-W4 cell expressing EGFP-UtrCH. Images were acquired every 30 seconds for 78 minutes; playback is 20 frames per second.Download Original Video (2.4 MB)https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e20-09-0582/20201005/media/mc-e20-09-0582-s04.,768,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518511abb28bed2c6f7421a565d9f7a5b3e3f5a40bc644191f5cacfb3cc9bc55304dc1c46558ab6522739decd45c6dec6a8654b8369cdf994bf5e08d6d62d0c63d92507c394aa0cf04dd46b92f24aee223a478de5bae27c9ab2716e1e04bcc229af0a816a872874585462c8102e260670350185913ddfa88ebe8e3087feb83c0c78878c28a944cd585fa92dcf5356bb9dec7ff927f293f434ff9a7Movie S5: Related to Figure 4. Microvillar core actin bundles are rapidly turned over. Spinning disk confocal imaging of a FRAP control experiment on induced Ls174T-W4 cell expressing mNeonGreen β-actin. ROI is bleached using a 405-laser line at 30% power with 100 μs dwell time. 5 frames acquired pre-bleach at 60 second intervals;22 frames acquired post-bleach at 30 second intervals for 10 minutes. Playback is 3 frames per second.Download Original Video (.6 MB)https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e20-09-0582/20201005/media/mc-e20-09-0582-s05.,768,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518511abb28bed2c6f7421a565d9f7a5b3e3f5a40bc644191f5cacfb3cc9bc55304dc1c46558ab6522739decd45c6dec6a8654b8369cdf994bf5e08d6d62d0c63d92507c394aa0cf04dd46b92f24aee223a478de5bae27c9ab2716e1e04bcc229af0a816a872874585462c8102e261670350185913dd48fc08141513cca21c947546745ff66939972a72a24aaaf8f15bbd134dce40a820c7ac5bb9Movie S6: Related to Figure 4. Inhibition of NM2 limits actin turnover. Spinning disk confocal imaging of a FRAP experiment on induced Ls174T-W4 cell expressing mNeonGreen-β-actin, treated with blebbistatin for 15 minutes prior to imaging. ROI is bleached using a 405-laser line at 30% power with 100 μs dwell time. 5 frames acquired pre-bleach at 60 second intervals; 22 frames acquired post-bleach at 30 second intervals for 10 minutes. Playback is 3 frames per second.Download Original Video (.7 MB)https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e20-09-0582/20201005/media/mc-e20-09-0582-s06.,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518515abb28bda92fbabe7b929571bd415190bf44d1790b7324fb90f184f91fab10e395327a1bef9725d0c99a358bbbe69c082a95f9d49957eb2932b2be9a6f328f5ec3ab3a0b9709f37f41cd72fbbabd2c3588c7488038615f2c4a4dbadb9df4030e588e612a3ac153bec21ed0aef5bbc6f1579cafa311d779d17731943b024dfc689b3d8ceed65bd41d2295581c3c8bb97f5912e99c2