Actin Dynamics Drive Microvillar Motility and Clustering during Brush Border Assembly.

LEAD CONTACT AND MATERIALS AVAILABILITY

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Matthew J. Tyska ( matthew.tyska@vanderbilt.edu ). EXPERIMENTAL MODEL AND SUBJECT DETAILS Cell Culture LLC-PK 1 -CL4 (CL4), CACO-2 BBE , and HEK293FT cells were cultured at 37°C and 5% CO 2 in DMEM with high glucose and 2 mM L-glutamine supplemented with 10% fetal bovine serum (FBS) except for CACO-2BBE cells which were supplemented with 20% FBS.

METHOD DETAILS Constructs

The pmCherry-Espin construct was a kind gift from Dr. James Bartles. pEGFP-Espin was generated by replacing mCherry with EGFP. The pGL-GPI-GFP was provided by the Dr. Anne Kenworthy (University of Virginia). The pEGFP-Myo1a as described in ( Tyska and Mooseker, 2002 ). The EGFP-Lifeact construct was provided by Dr. Irina Kaverina (Vanderbilt University). The pEGFP-NmMyo2c was purchased from Addgene, plasmid #10843. The mNEON-green-β-actin was purchased from Allele Biotechnology. The pEGFP-C1-IRTKS, pEGFP-C1-IRTKSDWH2, and pEGFP-C1-EPS8 as described in ( Postema et al., 2018 ). The pEGFP-C1-EPS8ΔAB construct (amino acids 1-648) was generated via PCR using EGFP-C1-EPS8 as a template. The PCR product was TOPO cloned into the pCR8/GW/TOPO vector (Invitrogen), and then shuttled into the EGFP-C1 backbone (Clontech), adapted for Gateway cloning using the Gateway conversion kit (Invitrogen). CDHR2-EGFP (PCDH24-EGFP as described in ( Crawley et al., 2014b ).

Stable Cell Line Generation

For generation of stable cell lines CL4 cells were grown to 80-90% confluency in T25 flasks and transfections were performed using Lipofectamine 2000 (Invitrogen) or FuGENE 6 (Promega) according to the manufacturer’s instructions. Selection for stable expression was performed after cells recovered for 2-3 days with addition of 1 mg/ml G418. For cells stably expressing two fluorescently-tagged proteins, the first construct was selected by transient transfection followed by antibiotic selection with G418 (e.g. EGFP-IRTKS, EGFP-IRTKSΔWH2, EGFP-EPS8, and EGFP-EPS8ΔAB). The second construct (mCherry-Espin) was introduced via viral transduction. Lentivirus was generated by co-transfecting HEK293FT cells (Fetal Hs embryonic epithelial cells; T75 flasks at 80% confluency) with 6 mg of pLVX-mCherry-Espin, 4 mg of psPAX2 packaging plasmid, and 0.8 mg of pMD2.G envelope plasmid using Lipofectamine 2000 (Invitrogen). For efficient lentiviral production, cells were incubated for 48 hours, then lentivirus-containing media was collected and concentrated with Lenti-X concentrator (Clontech). To transduce CL4 cells with lentivirus, the media was supplemented with 6 μg/ml polybrene (Sigma) and the lentiviral plasmid. After a 24-hour incubation, the media was changed and supplemented with 6 μg/ml polybrene and lentiviral plasmid for an additional 24 hours. Approximately 72 hours after initial viral transduction, cells were placed under antibiotic selection with Puromycin. Light Microscopy and Image Processing For SIM imaging, cells were plated on glass coverslips and allowed to grow to confluence. Cells were washed with warmed phosphate-buffered saline (PBS) and fixed with warm 4% paraformaldehyde/PBS for 15 min at 37°C. Cells were then washed three times with PBS, and blocked for 1 hour at room temperature in 5% bovine serum albumin (BSA) in PBS. Alexa Fluor 568-phalloidin (1:200, A12380; Invitrogen) or WGA-488 (10 μg/ml; W11261 ThermoFischer Scientific) were diluted in blocking solution and incubated for 1 hour at room temperature. Coverslips were washed three times with PBS then mounted on glass slides in ProLong Gold ( P36930 ; Invitrogen). Cells were imaged on a Nikon Structured Illumination Microscope (N-SIM), Andor DU-897 EMCCD camera with four color excitation lasers (405 nm, 488 nm, 561 nm, and 647 nm), and a 100x/1.49 NA TIRF objective. SIM images were reconstructed using the Nikon Elements reconstruction algorithm. For live-cell SDCM, cells were plated onto plasma-cleaned 35 mm glass bottom dishes (Invitro Scientific, D35-20-1.5-N), then transfected with the appropriate marker construct. Cells were allowed to grow to the appropriate level of confluence. If transfected, cells were imaged within 24 to 72 hours of transfection. Live-cell imaging was performed on a Nikon Ti2 inverted light microscope equipped with a Yokogawa CSU-X1 spinning disk head, Andor DU-897 EMCCD camera or a Photometrics Prime 95B sCMOS camera, 488 nm and 561 nm excitation lasers, a 405 nm photo-stimulation laser directed by a Bruker mini-scanner to enable targeted photoactivation, photoconversion, and photobleaching), and a 100x/1.49 NA TIRF objective. Low density microvilli were imaged when cells were subconfluent (80-90% confluence) or 1 DPC and images were acquired every 30-60 seconds for 20-40 minutes ( Figures 1 and 2 , and Supplemental Figure 2 ). During drug treatment, either (−)-Blebbistatin (B592500 Toronto Research Chemicals), Cythochalasin D (C2618 Sigma), or Cytochalasin B (C6762 Sigma) was added after 5 minutes of baseline measurement ( Figure 2 ). For photokinetic studies of actin dynamics, baseline images were obtained for several frames prior to bleaching, then for an additional 3-5 minutes of recovery at 10 second intervals. Bleaching was performed on a line ROI (0.1 μm in width and 3-15 μm in length) using a 405 nm laser at 30% power with a 10 μs dwell time. Higher density microvilli on cells 2 DPC were imaged every 1-2 minutes for up to 4 hours ( Figure 4 ). During imaging, cells were maintained with humidity at 37°C with 5% CO2 using a stage-top incubation system. Image acquisition was controlled with Nikon Elements software. 3D time series images were oversampled in the z-dimension with z-steps ranging from 0.09 μm to 0.18 μm followed by deconvolution (Nikon Elements Automatic or Richardson-Lucy algorithms) for better object resolution. Images were contrast enhanced, cropped, and aligned using Image J software (NIH), Nikon Elements, or Imaris (BITPLANE). Two-dimensional images were viewed as a maximum intensity projection. Three-dimensional depth coding was completed using Nikon Elements with images viewed as alpha-blended 3D composite images. Imaris (BITPLANE) was used to create an initial surface representing the microvillar fluorescence signal ( Figure 3 ) then manually adjusted with fusion/fission of adjacent objects and manual microvillar tracking. For live-correlative SDCM, cells were imaged as above, then washed with warmed phosphate-buffered saline (PBS) and fixed with warm 4% paraformaldehyde/PBS for 15 min at 37°C. Cells were then washed three times with PBS, and blocked for 1 hour at room temperature in 5% bovine serum albumin (BSA) in PBS. Primary antibody HPA009081 (Sigma) was diluted 1:200 in PBS and incubated with cells at 37°C for 1 hour followed by four washes with PBS. Cells were then incubated with secondary donkey anti-rabbit Alexa Fluor 488 (2 mg/ml, A-21206; Invitrogen) diluted 1:200 at room temperature. Cells were washed four times with PBS then imaged by SDCM. For live-cell LLSM, a customized version of a lattice light sheet (LLS) microscope was designed and built based on the LLS system originally published by the Betzig group at Janelia Farm Research Campus (JFRC, HHMI) and in accordance with a research license agreement in place between institutions ( Chen et al., 2014 ). In addition to light path modifications to enhance stability, efficiency, and accessibility of the instrument, upgrades particularly relevant for these experiments include a 583nm fiber laser (MPB Communications, Inc.), to excite mCherry at 99% of peak absorbance, and a newer generation sensor (Flash4.0v3, Hamamatsu, Inc.), which together substantially decreased the excitation energy necessary for time lapse imaging. In all cases, a dithered square lattice was utilized for imaging through the use of a spatial light modulator (Fourth Dimension Displays) in combination with an annular mask position corresponding to an annulus with 0.55 outer, and 0.44 inner, numerical apertures. Acquisition of datasets was managed through LabView (National Instruments). Image stacks contained anywhere from 300-500 optical sections per time point, with a step size ranging from 200 nm-320 nm between planes. Camera exposure times ranged from 5 msec - 15 msec per plane, with 25 sec intervals between stacks. Postacquisition, images were deskewed and deconvolved using 10 iterations of Richardson-Lucy Deconvolution. These post-acquisition processing steps were accomplished through LLSpy (Python code developed by Talley Lambert, Harvard University)( Talley, 2019 ). LLSpy implements cudaDeconv (Rev102), which is CUDA-based deskew and deconvolution developed by the Betzig Lab (JFRC) by Lin Shao and Dan Milkie. Maximum intensity projections were generated from resulting datasets via FIJI (ImageJ) software ( Schindelin et al., 2012 ).

Electron Microscopy

All electron microscopy (EM) reagents were purchased from Electron Microscopy Sciences. To prepare samples for EM, cells were plated on glass coverslips (SEM) or plastic dishes (TEM), washed once with warm SEM buffer (0.1M HEPES, pH 7.3) supplemented with 2 mM CaCl 2 , then sequentially fixed for 1 hour at room temperature with 2.5% glutaraldehyde and 4% paraformaldehyde in SEM buffer supplemented with 2mM CaCl 2 , washed with SEM buffer, incubated in 1% tannic acid in SEM buffer for 1 hour, washed with ddH 2 O, incubated with 1% OsO4 in ddH 2 O for 1 hour, washed with ddH 2 O, incubated with 1% uranyl acetate in ddH 2 O for 30, then washed with ddH 2 O. Samples were dehydrated in a graded ethanol series. After dehydration, SEM samples were then dried using critical point drying and mounted on aluminum stubs and coated with gold/palladium using a sputter coater. SEM imaging was performed using Quanta 250 Environmental-SEM operated in high vacuum mode with an accelerating voltage of 5-10 kV. After dehydration TEM samples were transitioned to propylene oxide and gradually infiltrated with a Quetol 651 formulation Spurr’s resin ( Ellis, 2006 ) using 1/4 th the reported amount of BDMA to reduce viscosity. The resin was polymerized for 48 hours at 60Ό until blocks were firm, but not brittle. Thin sections were cut at nominal thickness of 70 nm and poststained with 2% uranyl acetate and Reynold’s lead citrate. Images were collected with an FEI Technai T-12 transmission electron microscope operating at 100 kV using an AMT CCD camera.

QUANTIFICATION AND STATISTICAL ANALYSIS

Microvillar centroids (x and y coordinates, length, and angle) were manually tracked using ImageJ, and the tip position was calculated using Microsoft Excel ( Figure 1F , 2B – C , 2G – H , and Supplemental Figure 2F ). The rate of microvillar motility was calculated as the net microvillar tip movement over microvillar lifetime. Maximum microvillar length is the longest length measured for a microvillus during its lifetime. Persistence is calculated as the net microvillar tip displacement divided by the total path traveled. If tips move in a straight line, persistence is equal to 1. All calculations on drug treated samples use the time of drug addition as time = 0. Mean square displacement (MSD) and velocity autocorrelation analysis were completed on microvillar centroids tracked in ImageJ. Data was exported and analyzed with MATLAB using a package specifically developed for MSD analysis ( Tarantino et al., 2014 ), which is publicly available at http://www.mathworks.com/matlabcentral/fileexchange/40692-mean-square-displacement-analysis-of-particles-trajectories . For each individual trajectory, the MSD as a function of time delay (seconds) was calculated using Σ t ∣ → r ( t + D e l a y ) − → r ( t ) ∣ 2 and then averaged overall all trajectories. Random motion is indicated by a linear relationship whereas active motility is represented by convex curvature, and restricted motion by concave curvature. Active motility was fit to MSD ( n ) = 4 Dn + V 2 n 2 , where D is the diffusion coefficient, V is the velocity, and n is the time window used for calculation. For MSD plots, open circles represent the mean MSD, error bars are the SEM, and shaded areas represent the weighted SD over all MSD curves. The velocity autocorrelation is likewise calculated for each individual track and then averaged over trajectories. For random motion, displacements are not correlated, thus velocity autocorrelation is 0 except for the initial positive displacement (normalized to 1). For active motion, non-zero velocity autocorrelation is expected. For all figures, error bars indicate SD, and n values are reported in the figure legends. Statistical significance was calculated using the Mann-Whitney-Wilcoxon test for pairwise comparisons ( Figure 1I – K ), or using one-way ANOVA with Kruskal-Wallis to compare more than 2 conditions ( Figure 2K – M , Figure 4E – G ). Rose plots and individual tracks were graphed using Excel. MSD and normalized velocity autocorrelation were analyzed and graphed using MATLAB. All other graphs were generated in Prism v.7 (GraphPad). All statistical testing was performed in Prism v.7 (GraphPad) or MATLAB.

DATA AND CODE AVAILABILITY

No large-scale datasets or new code were generated in this study.

LEAD CONTACT AND MATERIALS AVAILABILITY

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Matthew J. Tyska ( matthew.tyska@vanderbilt.edu ).

EXPERIMENTAL MODEL AND SUBJECT DETAILS Cell Culture LLC-PK 1 -CL4 (CL4), CACO-2 BBE , and HEK293FT cells were cultured at 37°C and 5% CO 2 in DMEM with high glucose and 2 mM L-glutamine supplemented with 10% fetal bovine serum (FBS) except for CACO-2BBE cells which were supplemented with 20% FBS.

METHOD DETAILS Constructs

The pmCherry-Espin construct was a kind gift from Dr. James Bartles. pEGFP-Espin was generated by replacing mCherry with EGFP. The pGL-GPI-GFP was provided by the Dr. Anne Kenworthy (University of Virginia). The pEGFP-Myo1a as described in ( Tyska and Mooseker, 2002 ). The EGFP-Lifeact construct was provided by Dr. Irina Kaverina (Vanderbilt University). The pEGFP-NmMyo2c was purchased from Addgene, plasmid #10843. The mNEON-green-β-actin was purchased from Allele Biotechnology. The pEGFP-C1-IRTKS, pEGFP-C1-IRTKSDWH2, and pEGFP-C1-EPS8 as described in ( Postema et al., 2018 ). The pEGFP-C1-EPS8ΔAB construct (amino acids 1-648) was generated via PCR using EGFP-C1-EPS8 as a template. The PCR product was TOPO cloned into the pCR8/GW/TOPO vector (Invitrogen), and then shuttled into the EGFP-C1 backbone (Clontech), adapted for Gateway cloning using the Gateway conversion kit (Invitrogen). CDHR2-EGFP (PCDH24-EGFP as described in ( Crawley et al., 2014b ).

Stable Cell Line Generation

For generation of stable cell lines CL4 cells were grown to 80-90% confluency in T25 flasks and transfections were performed using Lipofectamine 2000 (Invitrogen) or FuGENE 6 (Promega) according to the manufacturer’s instructions. Selection for stable expression was performed after cells recovered for 2-3 days with addition of 1 mg/ml G418. For cells stably expressing two fluorescently-tagged proteins, the first construct was selected by transient transfection followed by antibiotic selection with G418 (e.g. EGFP-IRTKS, EGFP-IRTKSΔWH2, EGFP-EPS8, and EGFP-EPS8ΔAB). The second construct (mCherry-Espin) was introduced via viral transduction. Lentivirus was generated by co-transfecting HEK293FT cells (Fetal Hs embryonic epithelial cells; T75 flasks at 80% confluency) with 6 mg of pLVX-mCherry-Espin, 4 mg of psPAX2 packaging plasmid, and 0.8 mg of pMD2.G envelope plasmid using Lipofectamine 2000 (Invitrogen). For efficient lentiviral production, cells were incubated for 48 hours, then lentivirus-containing media was collected and concentrated with Lenti-X concentrator (Clontech). To transduce CL4 cells with lentivirus, the media was supplemented with 6 μg/ml polybrene (Sigma) and the lentiviral plasmid. After a 24-hour incubation, the media was changed and supplemented with 6 μg/ml polybrene and lentiviral plasmid for an additional 24 hours. Approximately 72 hours after initial viral transduction, cells were placed under antibiotic selection with Puromycin. Light Microscopy and Image Processing For SIM imaging, cells were plated on glass coverslips and allowed to grow to confluence. Cells were washed with warmed phosphate-buffered saline (PBS) and fixed with warm 4% paraformaldehyde/PBS for 15 min at 37°C. Cells were then washed three times with PBS, and blocked for 1 hour at room temperature in 5% bovine serum albumin (BSA) in PBS. Alexa Fluor 568-phalloidin (1:200, A12380; Invitrogen) or WGA-488 (10 μg/ml; W11261 ThermoFischer Scientific) were diluted in blocking solution and incubated for 1 hour at room temperature. Coverslips were washed three times with PBS then mounted on glass slides in ProLong Gold ( P36930 ; Invitrogen). Cells were imaged on a Nikon Structured Illumination Microscope (N-SIM), Andor DU-897 EMCCD camera with four color excitation lasers (405 nm, 488 nm, 561 nm, and 647 nm), and a 100x/1.49 NA TIRF objective. SIM images were reconstructed using the Nikon Elements reconstruction algorithm. For live-cell SDCM, cells were plated onto plasma-cleaned 35 mm glass bottom dishes (Invitro Scientific, D35-20-1.5-N), then transfected with the appropriate marker construct. Cells were allowed to grow to the appropriate level of confluence. If transfected, cells were imaged within 24 to 72 hours of transfection. Live-cell imaging was performed on a Nikon Ti2 inverted light microscope equipped with a Yokogawa CSU-X1 spinning disk head, Andor DU-897 EMCCD camera or a Photometrics Prime 95B sCMOS camera, 488 nm and 561 nm excitation lasers, a 405 nm photo-stimulation laser directed by a Bruker mini-scanner to enable targeted photoactivation, photoconversion, and photobleaching), and a 100x/1.49 NA TIRF objective. Low density microvilli were imaged when cells were subconfluent (80-90% confluence) or 1 DPC and images were acquired every 30-60 seconds for 20-40 minutes ( Figures 1 and 2 , and Supplemental Figure 2 ). During drug treatment, either (−)-Blebbistatin (B592500 Toronto Research Chemicals), Cythochalasin D (C2618 Sigma), or Cytochalasin B (C6762 Sigma) was added after 5 minutes of baseline measurement ( Figure 2 ). For photokinetic studies of actin dynamics, baseline images were obtained for several frames prior to bleaching, then for an additional 3-5 minutes of recovery at 10 second intervals. Bleaching was performed on a line ROI (0.1 μm in width and 3-15 μm in length) using a 405 nm laser at 30% power with a 10 μs dwell time. Higher density microvilli on cells 2 DPC were imaged every 1-2 minutes for up to 4 hours ( Figure 4 ). During imaging, cells were maintained with humidity at 37°C with 5% CO2 using a stage-top incubation system. Image acquisition was controlled with Nikon Elements software. 3D time series images were oversampled in the z-dimension with z-steps ranging from 0.09 μm to 0.18 μm followed by deconvolution (Nikon Elements Automatic or Richardson-Lucy algorithms) for better object resolution. Images were contrast enhanced, cropped, and aligned using Image J software (NIH), Nikon Elements, or Imaris (BITPLANE). Two-dimensional images were viewed as a maximum intensity projection. Three-dimensional depth coding was completed using Nikon Elements with images viewed as alpha-blended 3D composite images. Imaris (BITPLANE) was used to create an initial surface representing the microvillar fluorescence signal ( Figure 3 ) then manually adjusted with fusion/fission of adjacent objects and manual microvillar tracking. For live-correlative SDCM, cells were imaged as above, then washed with warmed phosphate-buffered saline (PBS) and fixed with warm 4% paraformaldehyde/PBS for 15 min at 37°C. Cells were then washed three times with PBS, and blocked for 1 hour at room temperature in 5% bovine serum albumin (BSA) in PBS. Primary antibody HPA009081 (Sigma) was diluted 1:200 in PBS and incubated with cells at 37°C for 1 hour followed by four washes with PBS. Cells were then incubated with secondary donkey anti-rabbit Alexa Fluor 488 (2 mg/ml, A-21206; Invitrogen) diluted 1:200 at room temperature. Cells were washed four times with PBS then imaged by SDCM. For live-cell LLSM, a customized version of a lattice light sheet (LLS) microscope was designed and built based on the LLS system originally published by the Betzig group at Janelia Farm Research Campus (JFRC, HHMI) and in accordance with a research license agreement in place between institutions ( Chen et al., 2014 ). In addition to light path modifications to enhance stability, efficiency, and accessibility of the instrument, upgrades particularly relevant for these experiments include a 583nm fiber laser (MPB Communications, Inc.), to excite mCherry at 99% of peak absorbance, and a newer generation sensor (Flash4.0v3, Hamamatsu, Inc.), which together substantially decreased the excitation energy necessary for time lapse imaging. In all cases, a dithered square lattice was utilized for imaging through the use of a spatial light modulator (Fourth Dimension Displays) in combination with an annular mask position corresponding to an annulus with 0.55 outer, and 0.44 inner, numerical apertures. Acquisition of datasets was managed through LabView (National Instruments). Image stacks contained anywhere from 300-500 optical sections per time point, with a step size ranging from 200 nm-320 nm between planes. Camera exposure times ranged from 5 msec - 15 msec per plane, with 25 sec intervals between stacks. Postacquisition, images were deskewed and deconvolved using 10 iterations of Richardson-Lucy Deconvolution. These post-acquisition processing steps were accomplished through LLSpy (Python code developed by Talley Lambert, Harvard University)( Talley, 2019 ). LLSpy implements cudaDeconv (Rev102), which is CUDA-based deskew and deconvolution developed by the Betzig Lab (JFRC) by Lin Shao and Dan Milkie. Maximum intensity projections were generated from resulting datasets via FIJI (ImageJ) software ( Schindelin et al., 2012 ).

Electron Microscopy

All electron microscopy (EM) reagents were purchased from Electron Microscopy Sciences. To prepare samples for EM, cells were plated on glass coverslips (SEM) or plastic dishes (TEM), washed once with warm SEM buffer (0.1M HEPES, pH 7.3) supplemented with 2 mM CaCl 2 , then sequentially fixed for 1 hour at room temperature with 2.5% glutaraldehyde and 4% paraformaldehyde in SEM buffer supplemented with 2mM CaCl 2 , washed with SEM buffer, incubated in 1% tannic acid in SEM buffer for 1 hour, washed with ddH 2 O, incubated with 1% OsO4 in ddH 2 O for 1 hour, washed with ddH 2 O, incubated with 1% uranyl acetate in ddH 2 O for 30, then washed with ddH 2 O. Samples were dehydrated in a graded ethanol series. After dehydration, SEM samples were then dried using critical point drying and mounted on aluminum stubs and coated with gold/palladium using a sputter coater. SEM imaging was performed using Quanta 250 Environmental-SEM operated in high vacuum mode with an accelerating voltage of 5-10 kV. After dehydration TEM samples were transitioned to propylene oxide and gradually infiltrated with a Quetol 651 formulation Spurr’s resin ( Ellis, 2006 ) using 1/4 th the reported amount of BDMA to reduce viscosity. The resin was polymerized for 48 hours at 60Ό until blocks were firm, but not brittle. Thin sections were cut at nominal thickness of 70 nm and poststained with 2% uranyl acetate and Reynold’s lead citrate. Images were collected with an FEI Technai T-12 transmission electron microscope operating at 100 kV using an AMT CCD camera.

Supplementary Material 1 Supplemental Video 1. Microvilli move across the cell surface, Related to Figure 1A – B .

Lattice light-sheet microscopy

(LLSM) time lapse images of CL4 cell stably expressing mCherry-Espin (corresponds to Figure 1A ). Duration 28 minutes, 30 frames per second (fps). Scale bar is 10 μm. Intensity color code. 9 Supplemental Video 9. Peripheral clusters of microvilli are stable over up to 12 hours, Related to Figure 5H . Time lapse images of CL4 cells stably expressing mCherry-Espin. Images were deconvolved, processed for extended depth of field (EDF), and visualized as a maximum intensity z-projection with an intensity color code (pictured at left). Individual microvilli (magenta) translocate across the cell surface, merge into small clusters and either dissipate or merge into large clusters of microvilli (yellow/orange) at the cell periphery. These large clusters of microvilli appear to continuously remodel but remain stable over many hours. Duration 12 hours, 30 fps. Movie corresponds to Figure 5H . Scale bar is 10 μm. 10 2 Supplemental Video 2. Microvilli move across the cell surface independent of F-actin marker, Related to Figure 1C , E and Figure S1D – E . Time lapse images of CL4 cell stably expressing mCherry-Espin (left panel, corresponds to Figure 1C ), or EGFP-Lifeact (right panel, corresponds to Figure S1D ). Duration 20 minutes, 12 frames per second (fps). Color-coded arrowheads highlight individual translocating microvilli. Scale bars are 10 μm. 3 Supplemental Video 3. Early actin structures protruding from the cell surface are wrapped in membrane over time, Related to Figure S1A – B . Time lapse images of CL4 cell stably expressing mCherry-Espin (left panel) and GPI-GFP (right panel), corresponds to Figure S2A – B . The 3D volume was rotated so the microvillus of interest is central and moves vertically across the image, and a depth-code was applied. Z-axis depth color code bottom left with tick marks at 1 μm intervals. Movie contains 6 frames taken at 1 minute intervals. 4 Supplemental Video 4. Microvillar motility requires actin assembly but not myosin contractility, Related to Figure 2 . Time lapse images of CL4 cell stably expressing mCherry-Espin treated with 20 μM Blebbistatin (left panel, corresponds to Figure 2A ) or 500 nM Cytochalasin B (representative movie, corresponding Figure 2G – J analysis) with the addition of drug at 5 minutes. Duration 30 minutes, 12 fps. Scale bars are 10 μm. 5 Supplemental Video 5. Microvillar F-actin core treadmills during microvillar motility, Related to Figure 3 . Time lapse images of CL4 cell transiently expressing mNEON-Green-β-actin (top panel), microvillar surface created in Imaris (middle panel), isolated microvillus of interest (bottom panel). Images correspond to Figure 3B – D . Duration 120 seconds, 5 fps. Scale bars are 2 μm. 6 Supplemental Video 6. IRTKS and EPS8 modulate microvillar motility dependent on actin-binding, Related to Figure 4 . Time lapse images of CL4 cells stably expressing mCherry-Espin (magenta) plus either EGFP-IRTKS (top left), EGFP-IRTKSΔWH2 (top right), EGFP-EPS8 (bottom left), or EGFP-EPS8ΔAB (bottom right) in green. Images correspond to Figure 4A – D . Duration 20 minutes, 12 fps. Scale bars ar 5 μm. 7 Supplemental Video 7. Microvillar translocation drives intermicrovillar collision facilitating cluster formation, Related to Figure 5A . Time lapse images of CL4 cell stably expressing mCherry-Espin. Duration 120 minutes, 10 fps. Movie corresponds to Figure 5A . 8 Supplemental Video 8. Microvillar translocation drives collision and clustering of individual microvilli as well as movement of large clusters of microvilli across the cell surface, Related to Figure 5C – D . Time lapse images of CL4 cell stably expressing mCherry-Espin. Left Panel Individual microvilli collide and remain connected then translocate across the cell joining with larger clusters of microvilli. Single microvilli are marked by arrowheads, then asterisks once they coalesce with a larger cluster. Duration 120 minutes, 10 fps. Movie corresponds to Figure 5B , zoom of Supplemental Movie 7 . Right Panel A large cluster of microvilli (asterisk) moves across the cell surface traveling left then upward. Duration 120 minutes, 10 fps. Movie corresponds to Figure 5C , zoom of Supplemental Movie 7 .