all animal procedures were performed in accordance with recommendations in the arvo statement for the use of animals in ophthalmic and vision research , in the guide for the care and use of laboratory animals by the national institutes of health , and under an approved protocol from the institutional animal care and use committees at the scripps research institute . mixed - background tmod1 mice used in this study all contained a cardiac - restricted -myosin heavy chain ( mhc ) promoter - driven tmod1 transgene , as previously described . genotyping was as described , and for brevity , mouse genotypes are referred to as tmod1 and tmod1 . mixed - background mice carried an endogenous mutation in the bfsp2/cp49 gene leading to a loss of beaded intermediate filaments in the lens . we restored wild - type bfsp2/cp49 alleles to tmod1 mice by backcrossing with wild - type c57bl6 mice , as previously described . all mice used in this study were littermates that carried the mhc - tmod1 transgene and wild - type bsfp2/cp49 . regarding rabbit polyclonal primary antibodies , anti - pan - fimbrin was a generous gift from paul matsudaira ( national university of singapore ) , and anti - human tmod1 was prepared in our laboratory . with respect to mouse monoclonal primary antibodies , anti--actinin ( nonsarcomeric , actn1 ) was from sigma - aldrich corp . louis , mo , usa ) , anti - arp3 was from bd biosciences ( 612134 ; san jose , ca , usa ) , anti - ezrin from sigma - aldrich corp . rat monoclonal primary antibody anti - n - cadherin was a generous gift from dietmar vestweber ( max - planck - institute for molecular biomedicine ) . secondary antibodies were alexa-488conjugated goat anti - rabbit ( a11008 ; thermo fisher scientific , grand island , ny , usa ) , alexa-488conjugated goat anti - mouse ( 115 - 545 - 166 , minimal cross - reaction ; jackson immunoresearch , west grove , pa , usa ) , alexa-647conjugated goat anti - rat ( 112 - 605 - 167 , minimal cross - reaction ; jackson immunoresearch ) , and alexa-647conjugated goat anti - mouse igg ( a21236 ; thermo fisher scientific ) . rhodamine - phalloidin ( r415 , thermo fisher scientific ) was used to stain f - actin , and hoechst 33258 ( b2883 ; sigma - aldrich corp . ) three - month - old wild - type lenses were prepared for scanning electron microscopy ( sem ) as previously described . briefly , lenses were removed from enucleated mouse eyes and immersed in 2.5% glutaraldehyde 2% formaldehyde in 0.1 m sodium cacodylate buffer at room temperature for 30 minutes to 1 hour . after critical point drying , lenses were split into quarters along the anterior  posterior axis , yielding a freshly fractured surface that revealed a surface spanning a complete equatorial lens radius . imaging was conducted with a phillips xl30 tmp scanning electron microscope ( fei company , eindhoven , the netherlands ) . to observe interlocking protrusions and paddles along the short sides of fiber cells , freshly isolated lenses from 2-month - old tmod1 and tmod1 mice were fixed in 2.5% glutaraldehyde in 0.1 m sodium cacodylate buffer , ph 7.3 , at room temperature for 48 to 72 hours . each lens was properly oriented and fractured in an anterior  posterior orientation with a sharp razor blade to expose the longitudinal features of the fiber cell short sides along the anterior , equatorial , and posterior regions of the lens . lens halves were then postfixed in 1% aqueous oso4 for 1 hour at room temperature , dehydrated in graded ethanol , and dried in a samdri-795 critical point dryer ( tousimis , inc . , lens halves were mounted on specimen stubs and coated with gold / palladium in a hummer 6.2 sputter coater ( anatech , inc . micrographs were taken with a jeol 820 scanning electron microscope at 10 kv ( jeol , tokyo , japan ) . the lens nucleus was used as a reference to determine the location of images taken and to ensure that images were from comparable areas of different lenses ( i.e. , comparable regions were located based on measurements from the center outward ) . freshly enucleated eyes were collected from 6-week - old mice . a small opening made at the corneal  scleral junction facilitated fixative penetration . after fixation , samples were washed in pbs , cryoprotected in 30% sucrose , frozen in optimal cutting temperature ( oct ) medium ( sakura finetek , torrance , ca , usa ) , and stored at 80c until sectioning . frozen sections ( 12 m thick ) were collected with a leica cm1950 cryostat ( wetzlar , germany ) . prolong gold antifade reagent ( thermo fisher scientific ) was used to mount the slides , and images were collected using a zeiss lsm780 confocal microscope ( oberkochen , germany ) . frozen sections with cross - sectionally oriented fiber cells near the lens equator were identified based on the thickness of the lens epithelium . staining was repeated on three samples from different mice for each genotype , and representative data are shown . after fixation , lenses were cut into quarters using a sharp scalpel along the anterior  posterior axis . lens quarters were postfixed in 1% paraformaldehyde in pbs for 15 minutes at room temperature and then washed two times briefly with pbs . lens quarters were then permeabilized and blocked using 3% normal goat serum , 3% bovine serum albumin , and 0.3% then , samples were washed three times , 5 minutes per wash , with pbs . lens quarters were incubated in secondary antibodies diluted in blocking solution for 3 hours at room temperature , followed by washing ( four times , 5 minutes per wash ) with pbs . lens quarters were stored in prolong gold antifade reagent at 4c until imaging . images and z - stacks with 0.2-m steps were collected of single fiber cells or small bundles of fibers from lens quarters using a zeiss lsm780 confocal microscope . fiber cell morphology and cell location in relation to the bulk lens quarter were used to approximate the maturity of imaged fiber cells . staining was repeated on at least three lenses from three different mice for each genotype , and representative data are shown . z - stack confocal images were analyzed using volocity 6.3 ( perkinelmer , waltham , wa , usa ) . noise reduction using the fine filter was applied to all z - stacks in all channels . the extended focus function in volocity was used to flatten z - stacks into a single image . for fluorescence intensity heat maps , single optical sections from z - stacks were separated into individual channels ( 16-bit grayscale images ) , and then heat maps were generated in imagej using the heatmap histogram plug - in ( http://www.samuelpean.com/heatmap-histogram/ [ in the public domain ] ) . for tortuosity and cell neck width measurements , we evaluated single optical sections of single fibers stained with rhodamine - phalloidin to outline cell contours from 9 tmod1 and 9 tmod1 mice . using imagej , we located the fiber cell edges using the find edges function and used the wand tool ( mode : legacy ; tolerance : 5.0 ) to measure the length of one of the curved edges of each fiber cell in pixels . tortuosity was calculated as the contour length of the cell edge divided by the straight end - to - end distance along the fiber measured through the center of each fiber . cell neck width across each fiber at the concave region was measured using the line tool . the average , standard error , and statistical significance were calculated and plotted in excel ( microsoft , redmond , wa , usa ) . statistical significance was determined using the student 's t - test , and p values less than 0.01 were considered statistically significant . all animal procedures were performed in accordance with recommendations in the arvo statement for the use of animals in ophthalmic and vision research , in the guide for the care and use of laboratory animals by the national institutes of health , and under an approved protocol from the institutional animal care and use committees at the scripps research institute . mixed - background tmod1 mice used in this study all contained a cardiac - restricted -myosin heavy chain ( mhc ) promoter - driven tmod1 transgene , as previously described . genotyping was as described , and for brevity , mouse genotypes are referred to as tmod1 and tmod1 . mixed - background mice carried an endogenous mutation in the bfsp2/cp49 gene leading to a loss of beaded intermediate filaments in the lens . we restored wild - type bfsp2/cp49 alleles to tmod1 mice by backcrossing with wild - type c57bl6 mice , as previously described . all mice used in this study were littermates that carried the mhc - tmod1 transgene and wild - type bsfp2/cp49 . regarding rabbit polyclonal primary antibodies , anti - pan - fimbrin was a generous gift from paul matsudaira ( national university of singapore ) , and anti - human tmod1 was prepared in our laboratory . with respect to mouse monoclonal primary antibodies , anti--actinin ( nonsarcomeric , actn1 ) was from sigma - aldrich corp . louis , mo , usa ) , anti - arp3 was from bd biosciences ( 612134 ; san jose , ca , usa ) , anti - ezrin from sigma - aldrich corp . rat monoclonal primary antibody anti - n - cadherin was a generous gift from dietmar vestweber ( max - planck - institute for molecular biomedicine ) . secondary antibodies were alexa-488conjugated goat anti - rabbit ( a11008 ; thermo fisher scientific , grand island , ny , usa ) , alexa-488conjugated goat anti - mouse ( 115 - 545 - 166 , minimal cross - reaction ; jackson immunoresearch , west grove , pa , usa ) , alexa-647conjugated goat anti - rat ( 112 - 605 - 167 , minimal cross - reaction ; jackson immunoresearch ) , and alexa-647conjugated goat anti - mouse igg ( a21236 ; thermo fisher scientific ) . rhodamine - phalloidin ( r415 , thermo fisher scientific ) was used to stain f - actin , and hoechst 33258 ( b2883 ; sigma - aldrich corp . ) three - month - old wild - type lenses were prepared for scanning electron microscopy ( sem ) as previously described . briefly , lenses were removed from enucleated mouse eyes and immersed in 2.5% glutaraldehyde 2% formaldehyde in 0.1 m sodium cacodylate buffer at room temperature for 30 minutes to 1 hour . after critical point drying , lenses were split into quarters along the anterior  posterior axis , yielding a freshly fractured surface that revealed a surface spanning a complete equatorial lens radius . imaging was conducted with a phillips xl30 tmp scanning electron microscope ( fei company , eindhoven , the netherlands ) . to observe interlocking protrusions and paddles along the short sides of fiber cells , freshly isolated lenses from 2-month - old tmod1 and tmod1 mice were fixed in 2.5% glutaraldehyde in 0.1 m sodium cacodylate buffer , ph 7.3 , at room temperature for 48 to 72 hours . each lens was properly oriented and fractured in an anterior  posterior orientation with a sharp razor blade to expose the longitudinal features of the fiber cell short sides along the anterior , equatorial , and posterior regions of the lens . lens halves were then postfixed in 1% aqueous oso4 for 1 hour at room temperature , dehydrated in graded ethanol , and dried in a samdri-795 critical point dryer ( tousimis , inc . , rockville , md , usa ) . lens halves were mounted on specimen stubs and coated with gold / palladium in a hummer 6.2 sputter coater ( anatech , inc . micrographs were taken with a jeol 820 scanning electron microscope at 10 kv ( jeol , tokyo , japan ) . the lens nucleus was used as a reference to determine the location of images taken and to ensure that images were from comparable areas of different lenses ( i.e. , comparable regions were located based on measurements from the center outward ) . freshly enucleated eyes were collected from 6-week - old mice . a small opening made at the corneal  scleral junction facilitated fixative penetration . after fixation , samples were washed in pbs , cryoprotected in 30% sucrose , frozen in optimal cutting temperature ( oct ) medium ( sakura finetek , torrance , ca , usa ) , and stored at 80c until sectioning . frozen sections ( 12 m thick ) were collected with a leica cm1950 cryostat ( wetzlar , germany ) . prolong gold antifade reagent ( thermo fisher scientific ) was used to mount the slides , and images were collected using a zeiss lsm780 confocal microscope ( oberkochen , germany ) . frozen sections with cross - sectionally oriented fiber cells near the lens equator were identified based on the thickness of the lens epithelium . staining was repeated on three samples from different mice for each genotype , and representative data are shown . after fixation , lenses were cut into quarters using a sharp scalpel along the anterior  posterior axis . lens quarters were postfixed in 1% paraformaldehyde in pbs for 15 minutes at room temperature and then washed two times briefly with pbs . lens quarters were then permeabilized and blocked using 3% normal goat serum , 3% bovine serum albumin , and 0.3% triton x-100 in pbs for 1 hour at room temperature . after blocking , then , samples were washed three times , 5 minutes per wash , with pbs . lens quarters were incubated in secondary antibodies diluted in blocking solution for 3 hours at room temperature , followed by washing ( four times , 5 minutes per wash ) with pbs . images and z - stacks with 0.2-m steps were collected of single fiber cells or small bundles of fibers from lens quarters using a zeiss lsm780 confocal microscope . fiber cell morphology and cell location in relation to the bulk lens quarter were used to approximate the maturity of imaged fiber cells . staining was repeated on at least three lenses from three different mice for each genotype , and representative data are shown . z - stack confocal images were analyzed using volocity 6.3 ( perkinelmer , waltham , wa , usa ) . noise reduction using the fine filter was applied to all z - stacks in all channels . the extended focus function in volocity was used to flatten z - stacks into a single image . for fluorescence intensity heat maps , single optical sections from z - stacks were separated into individual channels ( 16-bit grayscale images ) , and then heat maps were generated in imagej using the heatmap histogram plug - in ( http://www.samuelpean.com/heatmap-histogram/ [ in the public domain ] ) . for tortuosity and cell neck width measurements , we evaluated single optical sections of single fibers stained with rhodamine - phalloidin to outline cell contours from 9 tmod1 and 9 tmod1 mice . using imagej , we located the fiber cell edges using the find edges function and used the wand tool ( mode : legacy ; tolerance : 5.0 ) to measure the length of one of the curved edges of each fiber cell in pixels . tortuosity was calculated as the contour length of the cell edge divided by the straight end - to - end distance along the fiber measured through the center of each fiber . cell neck width across each fiber at the concave region was measured using the line tool . three measurements of cell neck width were averaged for each fiber cell . the average , standard error , and statistical significance were calculated and plotted in excel ( microsoft , redmond , wa , usa ) . statistical significance was determined using the student 's t - test , and p values less than 0.01 were considered statistically significant . classic electron microscopy studies have described the complex interface between differentiating and maturing lens fiber cells characterized by specialized membrane interdigitations , which is shown here by sem ( figs . are straight with small protrusions along the short sides and balls and sockets in the middle of the broad sides ( fig . 1b , cells viewed from the short - side edge ) . as fiber cells differentiate , increased small protrusions appear along their short sides ( fig . 1c , top - down view of the broad sides ) , with mature fiber cells developing elaborate large interlocking paddles decorated with small protrusions ( fig . 1d , top - down view of the broad sides ) . in small bundles of tmod1 lens fibers fixed and stained with phalloidin , we observed that f - actin is enriched at the interfaces between cortical ( fig . f - actin appears along the membrane of fiber cells and is present in regions of large paddles as well as small protrusions , outlining cell contours , but the precise location of f - actin with respect to the paddles or protrusions is unclear due to the complex geometry of the membrane interfaces . for clarity , a cartoon of mature fiber cells indicating the morphologic features of mature fiber cells ( large paddles , small protrusions , valley between large paddles and base of small protrusions ) that will be discussed below is shown in figure 1h . ( a  d ) scanning electron microscopy ( sem ) at various depths in 3-month - old wild - type ( wt ) lenses . boxed regions in ( a ) indicate the approximate location where ( b  d ) higher - magnification images were obtained . ( b  d ) cortical newly formed fiber cells ( b ) are straight , with balls and sockets along the broad sides and small protrusions along the vertices . as fiber cells differentiate ( c ) , the cells remain straight and more small protrusions are formed along the cell vertices . mature lens fibers ( d ) form large interlocking paddle domains decorated by small protrusions along the cell vertices . single fibers are highlighted in green to show the changes in cell morphology as the fibers mature . note that ( d ) is a different orientation of the same fiber cell shown in figure 3b of blankenship et al . ( e  g ) confocal fluorescence microscopy of phalloidin staining of fiber cells in 6-week - old tmod1 lens fiber cell bundles ( located at depths comparable to those in [ b  d ] ) reveals that f - actin is enriched in large paddle domains and small interlocking protrusions at the vertices of fiber cells . ( h ) diagram ( not to scale ) of mature lens fiber cells with large paddles ( light blue shading ) , valleys between large paddles ( red lines ) , small protrusions ( green shading ) , and bases of small protrusions ( yellow lines ) along the short sides of the cell . scale bars : 50 m ( a ) ; 6 m ( b  d ) ; 4 m ( e  g ) . to better visualize individual fiber cell morphologies and f - actin organization at different stages of maturation located at different depths in the lens mouse lenses were microdissected , decapsulated , and fixed overnight at 4c ; and after fixation , lenses were cut into quarters and postfixed before immunostaining . single fiber cells as well as fiber cell bundles spontaneously detached from the bulk lens quarters during the immunostaining process , and segments up to 200 m long could be imaged by confocal microscopy . from our preparation , we observed three types of fiber cells : tightly apposed fiber cell bundles ( fig . 2 ) , and single lens fibers that are detached from neighboring cells ( figs . immunostaining of three neighboring fiber cell segments showed that f - actin is enriched in protrusions and paddles , and that the complex morphology of this ingenious 3d zipper is well preserved in these partially separated fibers ( fig . three - dimensional reconstruction of the lens fibers shows interlocking paddles and protrusions between adjacent cells ( fig . while difficult to visualize in these images , each fiber cell has three sets of coordinated paddles and protrusions along the short sides , corresponding to the three vertices located on the short sides . two - dimensional ( 2d ) projections of single xz planes reveal normal hexagonal fiber cell shapes with enriched f - actin along the short sides near vertices , and less intense f - actin staining along the broad sides ( figs . 2c ) shows smooth f - actin staining along the cell membrane , while the xz projection through a region with paddles ( fig . 2d ) reveals f - actin  rich regions of paddles and protrusions at their vertices ( red asterisk ) . this is analogous to the view seen in a typical equatorial cross section through mature lens fibers that have undergone denucleation , 150 to 200 m from the lens capsule ( fig . 2e , hexagonal cell body outlined with blue dashed lines and paddles and protrusions indicated by a red asterisk ) . an extended focus image of the flattened z - stack of the individual fiber cells clearly demonstrates the interlocking paddles and protrusions between adjacent cells ( fig . 2 g , 2h ) reveal slight gaps between the interlocking domains of adjacent cells ( green and white cells in fig . 2 g , pink and white cells in fig . in favorable views of a fiber cell edge , f - actin appears more highly enriched in the small protrusions compared to paddles ( fig . note that due to the 3d nature of these complex interdigitations , single optical planes do not show all of the protrusions and paddles along the short sides of each cell . our results indicate that our new immunostaining method for detached single fibers faithfully preserves fiber cell morphology observed in situ by sem ( fig . 1 ) , thus allowing detailed studies of the proteins required for the formation and/or stability of protrusions and paddles along fiber cell vertices . confocal fluorescence microscopy images ( 2d single optical planes , extended focus flattened z - stack , 3d reconstruction and cross section ) of f - actin in neighboring mature fiber cells in 6-week - old tmod1 lenses . 3d reconstruction of z - stacks through three neighboring fibers shows interlocking structures between adjacent cells ( a ) and coordinated paddle domains between cells of neighboring layers ( b ) ( red arrows ) . dashed boxes show the level where single xz planes in ( c , d ) are derived . a single 2d xz plane from the 3d reconstruction shows normal hexagonal fiber cell morphology ( c , d ) ( hexagonal cell body outlined with blue dashed lines ) with paddles and protrusions along the short side ( d ) ( red asterisk ) . the f - actin staining pattern in dissociated fiber cells is similar to what is typically observed in cross sections through hexagonal mature lens fibers that have undergone denucleation ( e ) ( hexagonal cell body outlined with blue dashed lines ) , demonstrating enriched f - actin along the short sides of these fibers and in regions of paddles and protrusions ( red asterisk ) with less intense f - actin signals along the broad sides . extended focus image of the flattened z - stack shows f - actin  rich interlocking paddles decorated by small protrusions along the short sides of the cells , creating a 3d zipper between adjacent cells ( f ) . single xy optical sections along the anterior  posterior axis through the fiber cells ( g , h ) , where the dashed lines are drawn through the xz plane in ( c ) show interlocking domains between fibers with some separation between the cells . ( 2d , single optical plane ) of f - actin in fiber cells at various depths and sem in 6-week - old ( a ) and 2-month - old ( c ) tmod1 and tmod1 lenses . ( a ) diagrams of normal cortical , differentiating , and mature fiber cells are shown along the top . tmod1 and tmod1 cortical and differentiating fiber cells are straight with small f - actin protrusions along their vertices . tmod1 mature fibers have large paddle domains ( asterisks ) with f - actin  rich small protrusions , while tmod1 fiber cells have f - actin  positive protrusions , but very few paddles . ( b ) tmod1 mature fiber cells display a significant decrease in tortuosity ( n = 9 , * p < 0.01 ) , but the cell neck width was unaffected ( double - headed arrows in [ a ] ) between tmod1 and tmod1 lenses ( n = 9 ) . ( c ) low - magnification sem with boxed regions indicating the location of higher - magnification images . comparable regions are identified by measuring outward from the nucleus at the center of the lens , since some peripheral cortical fibers are lost in the sample preparation procedure . tmod1 lenses have rows of mature fiber cells with coordinated paddle protrusions that are decorated by smaller protrusions of equal size and spacing . scale bars : 4 m ( a ) ; 1 mm ( c ) ( low magnification left ) ; 4 m ( c ) ( high magnification ) . to investigate whether the tmod1-stablized actin cytoskeleton plays a role in the formation or stabilization of fiber cell interdigitations , we compared fiber cell morphologies and f - actin organization in single fiber cells from tmod1 and tmod1 lenses . we distinguished cortical , differentiating , and mature fiber segments by their unique morphologies ( diagram in fig . 3a ) and by observing the location of the single fiber with respect to depth in the bulk lens quarter . single optical sections reveal that f - actin is highly enriched at the fiber cell membrane and in small protrusions in all fiber cells from both tmod1 and tmod1 lenses , including cortical , differentiating , and mature single fibers ( fig . tmod1 and tmod1 cortical and differentiating fibers are straight with numerous small protrusions along their vertices , and no obvious differences in f - actin or in cell morphologies are observed . protrusions along cortical fibers appear finger - like , while protrusions along differentiating fibers have more obvious head and neck regions ( fig . the tmod1 mature fiber cell , which has undergone denucleation ( 150250 m deep from the lens capsule ) , displays large paddle domains with small protrusions ( fig . unlike cortical and differentiating fibers , which are not affected by absence of tmod1 , the tmod1 mature fiber cell lacks normal paddles despite retaining an overall undulating shape with abundant f - actin  rich small protrusions . a useful metric to compare paddle formation between control and knockout mature fiber cells is the contour length of the fiber cell edge , or tortuosity , measured along the paddle contours relative to the overall fiber cell length . indeed , tmod1 mature fiber cells had a statistically significant decrease in tortuosity compared to tmod1 mature fiber cells , consistent with a partial loss of large paddles ( fig . 3b ) to ensure that cells from a similar depth were being compared between tmod1 and tmod1 lenses and showed that there was no difference in the cell neck width . to validate the observations based on single fiber cell immunostaining , we visualized fiber cell edges by classic sem of half lenses to determine whether the loss of tmod1 affects lens fiber cell interdigitations and protrusion morphologies . this showed that tmod1 mature lens fibers are characterized by coordinated large paddle domains decorated by evenly spaced and sized small protrusions ( 0.51.5 m ) , as expected ( fig . 3c ) . similar to immunostained single fiber cells , the tmod1 mature lens fibers appear to have reduced paddles with irregularly sized and arranged smaller protrusions ( fig . 3c ) , although the shapes of whole individual fiber cells are difficult to assess in these sem preparations . the ability to immunostain and visualize individual mature lens fiber cells provides an opportunity to investigate the actin cytoskeleton composition of protrusions versus paddles , and how loss of tmod1 and perturbations of actin - associated proteins could account for defective paddles but not small protrusions in tmod1 mature fiber cells . we hypothesized that tmod1 and a subset of actin - binding proteins ( abps ) would be associated with paddles but not small protrusions , and that loss of tmod1 would affect paddle - associated abps but not protrusion - associated abps , illuminating mechanisms underlying paddle formation . first , we investigated locations of tmod1 and 2-spectrin in tmod1 mature lens fiber cells as compared to tmod1 mature lens fibers by immunostaining and confocal microscopy . z - stacks collected through single fibers were flattened to view and compare the morphology of tmod1 versus tmod1 cells ( fig . , we observed that both tmod1 and 2-spectrin were excluded from the small protrusions and instead tended to be located along the contours of the paddle domains , where both tmod1 and 2-spectrin staining signals appeared in large bright puncta . we also observed small bright 2-spectrin puncta along the membrane of the broad sides ( fig . 4a , center of cell ) , consistent with lens cross - section staining data in our previous study . in single optical sections through the cytoplasm of a tmod1 mature fiber , bright tmod1 and 2-spectrin puncta are located mainly in valleys between large paddle domains along the cell edges ( i.e. , regions of concave curvature ) ( figs . 4b , 4d , arrows ) . in contrast , in the tmod1 mature fiber cell with aberrant and attenuated paddle contours , the bright 2-spectrin puncta along cell edges are reduced in number and now mostly dispersed in the fiber cell cytoplasm , as seen in single optical sections through the middle of the cell ( fig . a heat map of 2-spectrin fluorescence intensity shows increased cytoplasmic staining signals in the knockout mature fiber cell ( fig . however , there were no obvious differences in the punctate 2-spectrin staining pattern along the broad sides of tmod1 cells as compared to tmod1 cells ( fig . 2-spectrin puncta are also occasionally observed at the base of the f - actin  rich small protrusions along the short sides ( at the vertices ) of the tmod1 and tmod1 fibers ( figs . no changes in overall f - actin staining intensity or distribution were observed , mostly likely because absence of tmod1 does not affect morphology of small protrusions where most of the f - actin is located ( fig . the presence of both tmod1 and 2-spectrin puncta in valleys between large paddles in tmod1 fiber cells supports the hypothesis that tmod1 plays a role in the formation or maintenance of large paddle domains by stabilizing a spectrin  actin network normally located in these domains in mature fiber cells . moreover , alterations in 2-spectrin puncta locations and distributions in tmod1 mature fibers are consistent with our previous results showing that the loss of tmod1 perturbs the spectrin network in lens fiber cells . immunostaining of single mature fiber cells from 6-week - old tmod1 and tmod1 lenses for tmod1 ( green ) , f - actin ( red ) , and 2-spectrin ( blue ) . the tmod1 fiber has f - actin  rich large paddle domains and small protrusions along cell vertices . while the tmod1 fiber has very few paddles , f - actin  rich small protrusions are still present . ( b , d ) single optical section ( 2d ) from a z - stack , showing a section through the cytoplasm of the fiber cells , with enlargements . tmod1 and 2-spectrin are enriched in puncta near the cell membrane in valleys between large paddle domains in the tmod1 fiber ( arrows ) , and 2-spectrin is also enriched at the base of small protrusions in tmod1 and tmod1 fibers ( arrowheads ) . the 2-spectrin staining signal appears diffuse and cytoplasmic ( asterisks ) with fewer membrane - associated puncta in the tmod1 fiber . ( c ) fluorescence intensity heat maps of 2-spectrin staining in tmod1 and tmod1 lens fibers show that 2-spectrin staining is more cytoplasmic ( asterisks ) in the tmod1 fiber . scale bars : 4 m ( a  c ) ; 2 m ( d ) . our new data show that tmod1 controls the morphology of large paddle domains in mature lens fibers , via stabilization of 2-spectrin associations at the base of paddle domains . these domains form the vertices between mature fiber cells visualized in lens cross sections and are associated with other abps , such as -actinin , arp3 , and ezrin . therefore , we investigated whether these abps are located in the small protrusions or large paddles and whether they are disrupted in tmod1 fibers . in mouse lenses , nonmuscle -actinin ( actn1 ) , a crosslinking protein for antiparallel actin filaments , has little or no staining signal in the cortical differentiating fibers , but is enriched at the short sides of mature fibers in a punctate pattern ( fig . bright tmod1 and -actinin puncta are colocalized in valleys between large paddles in the mature tmod1 lens fiber ( figs . 5b , 5c , arrows ) , while in the absence of tmod1 , -actinin is distributed discontinuously along the entire cell membrane , even in regions with the abnormal and attenuated paddles . careful examination of the -actinin  f - actin merged image reveals that the -actinin staining is now located at the bases of all the small protrusions ( fig . we performed western blotting on soluble and insoluble lens protein samples to determine whether there was an increase in -actinin protein levels in knockout lenses . we found no change in the amount of -actinin in tmod1 lenses compared to the control ( data not shown ) . these data suggest that in control fiber cells , tmod1 may restrict assembly of -actinin  f - actin antiparallel bundles to the valleys between large paddles , promoting paddle morphogenesis . by contrast , in the absence of tmod1 , there may be a global rearrangement of the f - actin , with increased -actinin on the membrane . ( a ) immunostaining of frozen lens section from 6-week - old tmod1 mice for -actinin ( green ) and f - actin ( red ) . the staining signal is enriched on the short sides of mature fiber cells with a punctate pattern . ( b , c ) immunostaining of single mature fiber cells ( 2d , single optical plane ) from 6-week - old tmod1 and tmod1 lenses for tmod1 ( green ) , f - actin ( red ) , and -actinin ( blue ) . similar to tmod1 and 2-spectrin , -actinin is enriched in large puncta in valleys between large paddle domains in the tmod1 fiber ( arrows in [ b , c ] ) . in the tmod1 fiber enlargement shows that -actinin is enriched near the base of small protrusions in the tmod1 mature fiber cell ( arrowheads in [ c ] ) . scale bars : 20 m ( a ) ; 4 m ( b ) ; 2 m ( c ) . next , we immunostained lenses for ezrin , which links f - actin networks to the plasma membrane and fimbrin ( aka plastin ) , a small crosslinker that bundles f - actin in a parallel orientation . similar to previous reports , ezrin was detected only in lens fibers , where it was enriched on the short sides of cortical fibers , especially at vertices , with more uniform membrane staining in mature lens fibers as visualized in cross sections ( fig . cross sections , fimbrin was present in epithelial cells and was also associated with the membranes of both differentiating and mature fiber cells ( fig . ezrin staining was continuous and colocalized with f - actin along the cell membrane in both tmod1 and tmod1 mature lens fibers ( fig . ezrin also appeared to be enriched at the base of small protrusions as well as in the valleys between large paddles ( fig . fimbrin was restricted to puncta at the base of small protrusions in both control and knockout cells ( figs . 6c , 6d , arrows ) and was not enriched in the valleys between large paddles , unlike tmod1 , 2-spectrin , and -actinin . neither ezrin nor fimbrin displayed any significant changes in the absence of tmod1 , suggesting that these f - actin networks are independent of tmod1 . ( a ) immunostaining of frozen lens section from 6-week - old tmod1 mice for ezrin ( green ) and f - actin ( red ) . ezrin is present along the membranes of lens fiber cells and enriched at the vertices of cortical fibers and along the short sides of mature fiber cells . ( b ) immunostaining of frozen lens section from 6-week - old tmod1 mice for fimbrin ( green ) and f - actin ( red ) . ( c , d ) immunostaining of single mature fiber cells ( 2d , single optical plane ) from 6-week - old tmod1 and tmod1 lenses for fimbrin ( green ) , f - actin ( red ) , and ezrin ( blue ) . as expected , ezrin colocalizes with f - actin along the cell membrane and in interdigitations of tmod1 and tmod1 fibers . interestingly , fimbrin is enriched in puncta near the base of small interlocking protrusions in tmod1 and tmod1 fibers ( arrows in [ c , d ] ) . enlargements show ezrin enrichment ( arrowheads in [ d ] ) at the base of small protrusions . scale bars : 20 m ( a , b ) ; 4 m ( c ) ; 2 m ( d ) . immunostaining of single mature fiber cells ( 2d , single optical plane ) from 6-week - old tmod1 and tmod1 lenses for arp3 ( green ) , f - actin ( red ) , and n - cadherin ( blue ) . ( a ) n - cadherin is localized along the cell membrane in tmod1 and tmod1 fibers and appears enriched along the base of protrusions and valleys between large paddles . arp3 is enriched in puncta ( arrows ) near the base of small interlocking protrusions ( arrows ) in tmod1 and tmod1 fibers . arp3 is also found in valleys between large paddles in the tmod1 fiber ( arrowheads ) . ( b ) enlargements show that weak arp3 staining extends into small protrusions ( open triangles ) in tmod1 and tmod1 fibers . arp3 puncta at the base of small protrusions are often accompanied by enriched n - cadherin staining ( arrows ) . previous work hypothesized that arp2/3 may drive actin filament branching to push protrusions out of fiber cell membranes , while a pulling force on the membrane may be mediated by clathrin complexes along the membrane of the neighboring fiber . arp2/3 initiates branched actin filament assembly from preexisting filaments , and activation of arp2/3 by wasp / wave family members and cortactin drives cadherin - directed actin assembly . we previously demonstrated that arp3 was enriched at the vertices of cortical fiber cells visualized in cross sections where it colocalized with n - cadherin . however , arp3 localization was not investigated in mature fibers , and thus we localized arp3 and n - cadherin in single mature fibers . arp3 was enriched in puncta at the base of the small f - actin  rich protrusions in both control and knockout mature fibers ( fig . 7 , arrows ) , and arp3 puncta were also found in valleys between large paddles in the tmod1 fiber ( fig . low - intensity arp3 staining can also be observed to extend into and fill small protrusions in both control and knockout fibers ( fig . 7b , open triangles ) . n - cadherin staining signal was predominantly at the cell membrane with a discontinuous pattern in control and knockout cells ( figs . 7a , 7b ) . this staining pattern is consistent with a recent study showing n - cadherin staining at the membranes in lens fiber cell bundles . we also observed that arp3 puncta at the base of small protrusions often coincide with areas of enriched n - cadherin staining ( fig . these observations would be consistent with the original hypothesis that arp3 and n - cadherin play a role in assembly of the small f - actin  rich protrusions in fiber cells . however , since loss of tmod1 does not greatly affect formation of small f - actin protrusions ( figs . 37 ) nor alter arp3 or n - cadherin localization , fiber cell f - actin networks regulated by arp3 or n - cadherin do not appear to be associated with the tmod1-regulated f - actin networks that control paddle domain morphology . classic electron microscopy studies have described the complex interface between differentiating and maturing lens fiber cells characterized by specialized membrane interdigitations , which is shown here by sem ( figs . are straight with small protrusions along the short sides and balls and sockets in the middle of the broad sides ( fig . 1b , cells viewed from the short - side edge ) . as fiber cells differentiate , increased small protrusions appear along their short sides ( fig . 1c , top - down view of the broad sides ) , with mature fiber cells developing elaborate large interlocking paddles decorated with small protrusions ( fig . 1d , top - down view of the broad sides ) . in small bundles of tmod1 lens fibers fixed and stained with phalloidin , we observed that f - actin is enriched at the interfaces between cortical ( fig . f - actin appears along the membrane of fiber cells and is present in regions of large paddles as well as small protrusions , outlining cell contours , but the precise location of f - actin with respect to the paddles or protrusions is unclear due to the complex geometry of the membrane interfaces . for clarity , a cartoon of mature fiber cells indicating the morphologic features of mature fiber cells ( large paddles , small protrusions , valley between large paddles and base of small protrusions ) that will be discussed below is shown in figure 1h . ( a  d ) scanning electron microscopy ( sem ) at various depths in 3-month - old wild - type ( wt ) lenses . boxed regions in ( a ) indicate the approximate location where ( b  d ) higher - magnification images were obtained . ( b  d ) cortical newly formed fiber cells ( b ) are straight , with balls and sockets along the broad sides and small protrusions along the vertices . as fiber cells differentiate ( c ) , the cells remain straight and more small protrusions are formed along the cell vertices . mature lens fibers ( d ) form large interlocking paddle domains decorated by small protrusions along the cell vertices . single fibers are highlighted in green to show the changes in cell morphology as the fibers mature . note that ( d ) is a different orientation of the same fiber cell shown in figure 3b of blankenship et al . ( e  g ) confocal fluorescence microscopy of phalloidin staining of fiber cells in 6-week - old tmod1 lens fiber cell bundles ( located at depths comparable to those in [ b  d ] ) reveals that f - actin is enriched in large paddle domains and small interlocking protrusions at the vertices of fiber cells . ( h ) diagram ( not to scale ) of mature lens fiber cells with large paddles ( light blue shading ) , valleys between large paddles ( red lines ) , small protrusions ( green shading ) , and bases of small protrusions ( yellow lines ) along the short sides of the cell . scale bars : 50 m ( a ) ; 6 m ( b  d ) ; 4 m ( e  g ) . to better visualize individual fiber cell morphologies and f - actin organization at different stages of maturation located at different depths in the lens mouse lenses were microdissected , decapsulated , and fixed overnight at 4c ; and after fixation , lenses were cut into quarters and postfixed before immunostaining . single fiber cells as well as fiber cell bundles spontaneously detached from the bulk lens quarters during the immunostaining process , and segments up to 200 m long could be imaged by confocal microscopy . from our preparation , we observed three types of fiber cells : tightly apposed fiber cell bundles ( fig . 2 ) , and single lens fibers that are detached from neighboring cells ( figs . immunostaining of three neighboring fiber cell segments showed that f - actin is enriched in protrusions and paddles , and that the complex morphology of this ingenious 3d zipper is well preserved in these partially separated fibers ( fig . three - dimensional reconstruction of the lens fibers shows interlocking paddles and protrusions between adjacent cells ( fig . while difficult to visualize in these images , each fiber cell has three sets of coordinated paddles and protrusions along the short sides , corresponding to the three vertices located on the short sides . two - dimensional ( 2d ) projections of single xz planes reveal normal hexagonal fiber cell shapes with enriched f - actin along the short sides near vertices , and less intense f - actin staining along the broad sides ( figs . 2c ) shows smooth f - actin staining along the cell membrane , while the xz projection through a region with paddles ( fig . 2d ) reveals f - actin  rich regions of paddles and protrusions at their vertices ( red asterisk ) . this is analogous to the view seen in a typical equatorial cross section through mature lens fibers that have undergone denucleation , 150 to 200 m from the lens capsule ( fig . 2e , hexagonal cell body outlined with blue dashed lines and paddles and protrusions indicated by a red asterisk ) . an extended focus image of the flattened z - stack of the individual fiber cells clearly demonstrates the interlocking paddles and protrusions between adjacent cells ( fig . 2 g , 2h ) reveal slight gaps between the interlocking domains of adjacent cells ( green and white cells in fig . 2 g , pink and white cells in fig . in favorable views of a fiber cell edge , f - actin appears more highly enriched in the small protrusions compared to paddles ( fig . note that due to the 3d nature of these complex interdigitations , single optical planes do not show all of the protrusions and paddles along the short sides of each cell . our results indicate that our new immunostaining method for detached single fibers faithfully preserves fiber cell morphology observed in situ by sem ( fig . 1 ) , thus allowing detailed studies of the proteins required for the formation and/or stability of protrusions and paddles along fiber cell vertices . confocal fluorescence microscopy images ( 2d single optical planes , extended focus flattened z - stack , 3d reconstruction and cross section ) of f - actin in neighboring mature fiber cells in 6-week - old tmod1 lenses . 3d reconstruction of z - stacks through three neighboring fibers shows interlocking structures between adjacent cells ( a ) and coordinated paddle domains between cells of neighboring layers ( b ) ( red arrows ) . dashed boxes show the level where single xz planes in ( c , d ) are derived . a single 2d xz plane from the 3d reconstruction shows normal hexagonal fiber cell morphology ( c , d ) ( hexagonal cell body outlined with blue dashed lines ) with paddles and protrusions along the short side ( d ) ( red asterisk ) . the f - actin staining pattern in dissociated fiber cells is similar to what is typically observed in cross sections through hexagonal mature lens fibers that have undergone denucleation ( e ) ( hexagonal cell body outlined with blue dashed lines ) , demonstrating enriched f - actin along the short sides of these fibers and in regions of paddles and protrusions ( red asterisk ) with less intense f - actin signals along the broad sides . extended focus image of the flattened z - stack shows f - actin  rich interlocking paddles decorated by small protrusions along the short sides of the cells , creating a 3d zipper between adjacent cells ( f ) . single xy optical sections along the anterior  posterior axis through the fiber cells ( g , h ) , where the dashed lines are drawn through the xz plane in ( c ) show interlocking domains between fibers with some separation between the cells . ( 2d , single optical plane ) of f - actin in fiber cells at various depths and sem in 6-week - old ( a ) and 2-month - old ( c ) tmod1 and tmod1 lenses . ( a ) diagrams of normal cortical , differentiating , and mature fiber cells are shown along the top . tmod1 and tmod1 cortical and differentiating fiber cells are straight with small f - actin protrusions along their vertices . tmod1 mature fibers have large paddle domains ( asterisks ) with f - actin  rich small protrusions , while tmod1 fiber cells have f - actin  positive protrusions , but very few paddles . ( b ) tmod1 mature fiber cells display a significant decrease in tortuosity ( n = 9 , * p < 0.01 ) , but the cell neck width was unaffected ( double - headed arrows in [ a ] ) between tmod1 and tmod1 lenses ( n = 9 ) . ( c ) low - magnification sem with boxed regions indicating the location of higher - magnification images . comparable regions are identified by measuring outward from the nucleus at the center of the lens , since some peripheral cortical fibers are lost in the sample preparation procedure . tmod1 lenses have rows of mature fiber cells with coordinated paddle protrusions that are decorated by smaller protrusions of equal size and spacing . scale bars : 4 m ( a ) ; 1 mm ( c ) ( low magnification left ) ; 4 m ( c ) ( high magnification ) . to investigate whether the tmod1-stablized actin cytoskeleton plays a role in the formation or stabilization of fiber cell interdigitations , we compared fiber cell morphologies and f - actin organization in single fiber cells from tmod1 and tmod1 lenses . we distinguished cortical , differentiating , and mature fiber segments by their unique morphologies ( diagram in fig . 3a ) and by observing the location of the single fiber with respect to depth in the bulk lens quarter . single optical sections reveal that f - actin is highly enriched at the fiber cell membrane and in small protrusions in all fiber cells from both tmod1 and tmod1 lenses , including cortical , differentiating , and mature single fibers ( fig . tmod1 and tmod1 cortical and differentiating fibers are straight with numerous small protrusions along their vertices , and no obvious differences in f - actin or in cell morphologies are observed . protrusions along cortical fibers appear finger - like , while protrusions along differentiating fibers have more obvious head and neck regions ( fig . the tmod1 mature fiber cell , which has undergone denucleation ( 150250 m deep from the lens capsule ) , displays large paddle domains with small protrusions ( fig . unlike cortical and differentiating fibers , which are not affected by absence of tmod1 , the tmod1 mature fiber cell lacks normal paddles despite retaining an overall undulating shape with abundant f - actin  rich small protrusions . a useful metric to compare paddle formation between control and knockout mature fiber cells is the contour length of the fiber cell edge , or tortuosity , measured along the paddle contours relative to the overall fiber cell length . indeed , tmod1 mature fiber cells had a statistically significant decrease in tortuosity compared to tmod1 mature fiber cells , consistent with a partial loss of large paddles ( fig . 3b ) to ensure that cells from a similar depth were being compared between tmod1 and tmod1 lenses and showed that there was no difference in the cell neck width . to validate the observations based on single fiber cell immunostaining , we visualized fiber cell edges by classic sem of half lenses to determine whether the loss of tmod1 affects lens fiber cell interdigitations and protrusion morphologies . this showed that tmod1 mature lens fibers are characterized by coordinated large paddle domains decorated by evenly spaced and sized small protrusions ( 0.51.5 m ) , as expected ( fig . 3c ) . similar to immunostained single fiber cells , the tmod1 mature lens fibers appear to have reduced paddles with irregularly sized and arranged smaller protrusions ( fig . 3c ) , although the shapes of whole individual fiber cells are difficult to assess in these sem preparations . the ability to immunostain and visualize individual mature lens fiber cells provides an opportunity to investigate the actin cytoskeleton composition of protrusions versus paddles , and how loss of tmod1 and perturbations of actin - associated proteins could account for defective paddles but not small protrusions in tmod1 mature fiber cells . we hypothesized that tmod1 and a subset of actin - binding proteins ( abps ) would be associated with paddles but not small protrusions , and that loss of tmod1 would affect paddle - associated abps but not protrusion - associated abps , illuminating mechanisms underlying paddle formation . first , we investigated locations of tmod1 and 2-spectrin in tmod1 mature lens fiber cells as compared to tmod1 mature lens fibers by immunostaining and confocal microscopy . z - stacks collected through single fibers were flattened to view and compare the morphology of tmod1 versus tmod1 cells ( fig . , we observed that both tmod1 and 2-spectrin were excluded from the small protrusions and instead tended to be located along the contours of the paddle domains , where both tmod1 and 2-spectrin staining signals appeared in large bright puncta . we also observed small bright 2-spectrin puncta along the membrane of the broad sides ( fig . 4a , center of cell ) , consistent with lens cross - section staining data in our previous study . in single optical sections through the cytoplasm of a tmod1 mature fiber , bright tmod1 and 2-spectrin puncta are located mainly in valleys between large paddle domains along the cell edges ( i.e. , regions of concave curvature ) ( figs . 4b , 4d , arrows ) . in contrast , in the tmod1 mature fiber cell with aberrant and attenuated paddle contours , the bright 2-spectrin puncta along cell edges are reduced in number and now mostly dispersed in the fiber cell cytoplasm , as seen in single optical sections through the middle of the cell ( fig . a heat map of 2-spectrin fluorescence intensity shows increased cytoplasmic staining signals in the knockout mature fiber cell ( fig . however , there were no obvious differences in the punctate 2-spectrin staining pattern along the broad sides of tmod1 cells as compared to tmod1 cells ( fig . 2-spectrin puncta are also occasionally observed at the base of the f - actin  rich small protrusions along the short sides ( at the vertices ) of the tmod1 and tmod1 fibers ( figs . no changes in overall f - actin staining intensity or distribution were observed , mostly likely because absence of tmod1 does not affect morphology of small protrusions where most of the f - actin is located ( fig . the presence of both tmod1 and 2-spectrin puncta in valleys between large paddles in tmod1 fiber cells supports the hypothesis that tmod1 plays a role in the formation or maintenance of large paddle domains by stabilizing a spectrin  actin network normally located in these domains in mature fiber cells . moreover , alterations in 2-spectrin puncta locations and distributions in tmod1 mature fibers are consistent with our previous results showing that the loss of tmod1 perturbs the spectrin network in lens fiber cells . immunostaining of single mature fiber cells from 6-week - old tmod1 and tmod1 lenses for tmod1 ( green ) , f - actin ( red ) , and 2-spectrin ( blue ) . the tmod1 fiber has f - actin  rich large paddle domains and small protrusions along cell vertices . while the tmod1 fiber has very few paddles , f - actin  rich small protrusions are still present . ( b , d ) single optical section ( 2d ) from a z - stack , showing a section through the cytoplasm of the fiber cells , with enlargements . tmod1 and 2-spectrin are enriched in puncta near the cell membrane in valleys between large paddle domains in the tmod1 fiber ( arrows ) , and 2-spectrin is also enriched at the base of small protrusions in tmod1 and tmod1 fibers ( arrowheads ) . the 2-spectrin staining signal appears diffuse and cytoplasmic ( asterisks ) with fewer membrane - associated puncta in the tmod1 fiber . ( c ) fluorescence intensity heat maps of 2-spectrin staining in tmod1 and tmod1 lens fibers show that 2-spectrin staining is more cytoplasmic ( asterisks ) in the tmod1 fiber . scale bars : 4 m ( a  c ) ; 2 m ( d ) . our new data show that tmod1 controls the morphology of large paddle domains in mature lens fibers , via stabilization of 2-spectrin associations at the base of paddle domains . these domains form the vertices between mature fiber cells visualized in lens cross sections and are associated with other abps , such as -actinin , arp3 , and ezrin . therefore , we investigated whether these abps are located in the small protrusions or large paddles and whether they are disrupted in tmod1 fibers . in mouse lenses , nonmuscle -actinin ( actn1 ) , a crosslinking protein for antiparallel actin filaments , has little or no staining signal in the cortical differentiating fibers , but is enriched at the short sides of mature fibers in a punctate pattern ( fig . bright tmod1 and -actinin puncta are colocalized in valleys between large paddles in the mature tmod1 lens fiber ( figs . 5b , 5c , arrows ) , while in the absence of tmod1 , -actinin is distributed discontinuously along the entire cell membrane , even in regions with the abnormal and attenuated paddles . careful examination of the -actinin  f - actin merged image reveals that the -actinin staining is now located at the bases of all the small protrusions ( fig . we performed western blotting on soluble and insoluble lens protein samples to determine whether there was an increase in -actinin protein levels in knockout lenses . we found no change in the amount of -actinin in tmod1 lenses compared to the control ( data not shown ) . these data suggest that in control fiber cells , tmod1 may restrict assembly of -actinin  f - actin antiparallel bundles to the valleys between large paddles , promoting paddle morphogenesis . by contrast , in the absence of tmod1 , there may be a global rearrangement of the f - actin , with increased -actinin on the membrane . ( a ) immunostaining of frozen lens section from 6-week - old tmod1 mice for -actinin ( green ) and f - actin ( red ) . the staining signal is enriched on the short sides of mature fiber cells with a punctate pattern . ( b , c ) immunostaining of single mature fiber cells ( 2d , single optical plane ) from 6-week - old tmod1 and tmod1 lenses for tmod1 ( green ) , f - actin ( red ) , and -actinin ( blue ) . similar to tmod1 and 2-spectrin , -actinin is enriched in large puncta in valleys between large paddle domains in the tmod1 fiber ( arrows in [ b , c ] ) . in the tmod1 fiber enlargement shows that -actinin is enriched near the base of small protrusions in the tmod1 mature fiber cell ( arrowheads in [ c ] ) . scale bars : 20 m ( a ) ; 4 m ( b ) ; 2 m ( c ) . next , we immunostained lenses for ezrin , which links f - actin networks to the plasma membrane and fimbrin ( aka plastin ) , a small crosslinker that bundles f - actin in a parallel orientation . similar to previous reports , ezrin was detected only in lens fibers , where it was enriched on the short sides of cortical fibers , especially at vertices , with more uniform membrane staining in mature lens fibers as visualized in cross sections ( fig . cross sections , fimbrin was present in epithelial cells and was also associated with the membranes of both differentiating and mature fiber cells ( fig . ezrin staining was continuous and colocalized with f - actin along the cell membrane in both tmod1 and tmod1 mature lens fibers ( fig . ezrin also appeared to be enriched at the base of small protrusions as well as in the valleys between large paddles ( fig . fimbrin was restricted to puncta at the base of small protrusions in both control and knockout cells ( figs . 6c , 6d , arrows ) and was not enriched in the valleys between large paddles , unlike tmod1 , 2-spectrin , and -actinin . neither ezrin nor fimbrin displayed any significant changes in the absence of tmod1 , suggesting that these f - actin networks are independent of tmod1 . ( a ) immunostaining of frozen lens section from 6-week - old tmod1 mice for ezrin ( green ) and f - actin ( red ) . ezrin is present along the membranes of lens fiber cells and enriched at the vertices of cortical fibers and along the short sides of mature fiber cells . ( b ) immunostaining of frozen lens section from 6-week - old tmod1 mice for fimbrin ( green ) and f - actin ( red ) . ( c , d ) immunostaining of single mature fiber cells ( 2d , single optical plane ) from 6-week - old tmod1 and tmod1 lenses for fimbrin ( green ) , f - actin ( red ) , and ezrin ( blue ) . as expected , ezrin colocalizes with f - actin along the cell membrane and in interdigitations of tmod1 and tmod1 fibers . interestingly , fimbrin is enriched in puncta near the base of small interlocking protrusions in tmod1 and tmod1 fibers ( arrows in [ c , d ] ) . enlargements show ezrin enrichment ( arrowheads in [ d ] ) at the base of small protrusions . scale bars : 20 m ( a , b ) ; 4 m ( c ) ; 2 m ( d ) . immunostaining of single mature fiber cells ( 2d , single optical plane ) from 6-week - old tmod1 and tmod1 lenses for arp3 ( green ) , f - actin ( red ) , and n - cadherin ( blue ) . ( a ) n - cadherin is localized along the cell membrane in tmod1 and tmod1 fibers and appears enriched along the base of protrusions and valleys between large paddles . arp3 is enriched in puncta ( arrows ) near the base of small interlocking protrusions ( arrows ) in tmod1 and tmod1 fibers . arp3 is also found in valleys between large paddles in the tmod1 fiber ( arrowheads ) . ( b ) enlargements show that weak arp3 staining extends into small protrusions ( open triangles ) in tmod1 and tmod1 fibers . arp3 puncta at the base of small protrusions are often accompanied by enriched n - cadherin staining ( arrows ) . previous work hypothesized that arp2/3 may drive actin filament branching to push protrusions out of fiber cell membranes , while a pulling force on the membrane may be mediated by clathrin complexes along the membrane of the neighboring fiber . arp2/3 initiates branched actin filament assembly from preexisting filaments , and activation of arp2/3 by wasp / wave family members and cortactin drives cadherin - directed actin assembly . we previously demonstrated that arp3 was enriched at the vertices of cortical fiber cells visualized in cross sections where it colocalized with n - cadherin . however , arp3 localization was not investigated in mature fibers , and thus we localized arp3 and n - cadherin in single mature fibers . arp3 was enriched in puncta at the base of the small f - actin  rich protrusions in both control and knockout mature fibers ( fig . 7 , arrows ) , and arp3 puncta were also found in valleys between large paddles in the tmod1 fiber ( fig . low - intensity arp3 staining can also be observed to extend into and fill small protrusions in both control and knockout fibers ( fig . n - cadherin staining signal was predominantly at the cell membrane with a discontinuous pattern in control and knockout cells ( figs . 7a , 7b ) . this staining pattern is consistent with a recent study showing n - cadherin staining at the membranes in lens fiber cell bundles . we also observed that arp3 puncta at the base of small protrusions often coincide with areas of enriched n - cadherin staining ( fig . these observations would be consistent with the original hypothesis that arp3 and n - cadherin play a role in assembly of the small f - actin  rich protrusions in fiber cells . however , since loss of tmod1 does not greatly affect formation of small f - actin protrusions ( figs . 37 ) nor alter arp3 or n - cadherin localization , fiber cell f - actin networks regulated by arp3 or n - cadherin do not appear to be associated with the tmod1-regulated f - actin networks that control paddle domain morphology . we have developed a novel technique to immunostain single lens fiber cells while preserving their complex morphology ; this new method has allowed us to conduct detailed investigations of the f - actin structures and abps required for the formation of the interdigitations at fiber cell vertices . our study has shown that tmod1 is required for the formation of large paddles , but not small protrusions , between mature lens fiber cells . these subtle changes in fiber cell morphology are correlated with decreased stiffness in tmod1 lenses at low mechanical loads , providing the first evidence for the hypothesis that fiber cell interdigitations influence lens mechanical integrity . by contrast , the tmod1 lenses with reduced paddle domains between mature lens fibers remain transparent , indicating that formation of large paddles is not required for lens transparency . while recent studies have suggested that aqp0 or ephrin - a5 may be important for normal fiber cell interdigitations , 
 aqp0 and mixed - background efna5 lenses have severe cataract phenotypes with fiber cell degeneration , which complicates interpretations and impedes further studies . fortunately , the very mild phenotype in tmod1 lenses has given us an opportunity to dissect the mechanisms underlying the formation of small protrusions as compared to large paddles located at the vertices of mature lens fiber cells . ( a ) during fiber cell maturation , large paddle domains with small interlocking protrusions form along the vertices of hexagonal fiber cells . ( b ) tmod1 may stabilize the f - actin  spectrin network as well as -actinin  crosslinked antiparallel f - actin bundles in valleys between large paddles to maintain their structure . formation of small protrusions may be facilitated by arp3-nucleated actin networks , and fimbrin - crosslinked parallel f - actin bundles at the base of protrusions , while ezrin may stabilize actin networks along the entire fiber cell membrane and n - cadherin promotes cell  cell interactions . our novel immunostaining protocol for single fiber cells has yielded a wealth of new information about the proteins that control f - actin organization in small protrusions and in large paddles at the vertices of lens fiber cells ( fig . actin network clearly play a key role in the formation and/or maintenance of the large paddle domains in mature lens fibers . abnormal cytosolic localization of 2-spectrin in knockout single fibers extends our previous work showing that loss of tmod1 leads to disruptions in the lens fiber membrane skeleton by providing a molecular mechanism connecting the f - actin cytoskeleton to fiber cell morphology . unlike loss of tmod1 alone , which leads to selective reduction of large paddle domains , we showed previously that combined loss of tmod1 and cp49 , a beaded intermediate filament protein , leads to reduction in small protrusions at the vertices of mature fiber cells , suggesting that both actin and intermediate filaments are needed for normal formation or stabilization of these smaller interdigitations . interestingly , -actinin is also found in foci in the valleys between large paddles where it colocalizes with tmod1 in mature tmod1 fiber cells , suggesting that tmod1 may cap the pointed ends of actin filaments crosslinked by spectrin and/or -actinin . -actinin belongs to a protein family that includes spectrin , dystrophin , and utrophin , and -actinin  crosslinked f - actin bundles can interact with myosin ii to generate contractile forces . it is possible that -actinin  bundled f - actin interacts with myosin ii to generate force to push and/or pull the fiber cell membrane to create the large paddles . strikingly , in tmod1 mature fiber cells , the -actinin staining signal becomes more broadly localized along the fiber cell membrane in between the small protrusions . however , similar to 2-spectrin , we observe changes in the localization of -actinin without changes in the protein level . this implies that -actinin may be restricted to tmod1-capped f - actin in wild - type cells but become aberrantly distributed and abnormally crosslink other f - actin populations in the absence of tmod1 . since tmod1 lenses have reduced levels of tropomyosin ( tm ) , a stabilizing protein that binds along the side of f - actin and enhances tmod1 capping of actin pointed ends , and tm inhibits -actinin binding to f - actin , additional f - actin binding sites may now be available in knockout lens fibers for -actinin binding . it may also be significant that the localization of -actinin in the tmod1 mature fiber cell resembles that of n - cadherin in the tmod1 mature fiber cell . this suggests the possibility that expansion of -actinin  crosslinked f - actin networks at n - cadherin  mediated cell cell junctions may partially compensate for loss of spectrin  actin networks , perhaps as a mechanical response , albeit being unable to assist fiber cells in forming large paddle domains . our data in the lens parallel abnormal -actinin and f - actin staining patterns in tmod1 embryonic heart muscle cells that fail to assemble myofibrils . further studies will be needed to define the interactions between tmod1 and -actinin  associated f - actin , as well as the changes in -actinin and actin binding in the absence of tmod1 . this work also provides the first evidence that distinct abps may selectively control the formation of small protrusions as compared to large paddles in lens fiber cells . in addition to intestinal microvilli , fimbrin has been found in protrusions in other cell types , including stereocilia of the inner ear hair cells and microvilli of photoreceptors , taste receptor cells , nasal epithelial cells , and respiratory brush cells . the location of fimbrin in puncta at the base , but not throughout the small f - actin  rich protrusions in single mature fiber cells , suggests that these structures are not analogous to the microvilli of other cells , where fimbrin is present along their entire length . instead , fimbrin crosslinking of f - actin into rigid parallel filament bundles may help in stabilizing the small protrusions at their bases . our data are also consistent with previous studies showing that the presence of fimbrin and -actinin is mutually exclusive due to the different spacings between f - actins when bundled by fimbrin or -actinin . while fimbrin is found in small puncta at the base of the small protrusions in mature fiber cells , ezrin is associated with f - actin along the entire membrane of mature fiber cells , including along the membranes of small protrusions . immunoprecipitation experiments have shown that an actin anchorage complex consisting of ezrin  periplakin  periaxin  desmoyokin ( eppd ) proteins is associated with moesin , spectrin , and plectin in the lens , suggesting an interaction between erm , eppd , and the spectrin  actin networks . decreased actin and spectrin protein levels and fiber cell membrane staining signals are observed in periaxin knockout lenses , further indicating that the eppd complexes may function to stabilize the spectrin our work strengthens the hypothesis that arp2/3 may drive the formation of small protrusions by nucleating branched f - actin networks . we had expected arp3 to be enriched with f - actin within the small protrusions ; instead , we found that arp3 is predominantly enriched in bright puncta at the base of small protrusions , although dim , diffuse arp3 staining can be detected extending into small protrusions . this suggests that arp2/3 nucleation of branched f - actin networks occurs predominantly near the cell membrane at the base of protrusions and paddles , and to a lesser extent within the body of the protrusion , as originally proposed . notably , location of arp3 at the base of small protrusions where n - cadherin is also located implies that arp2/3 may nucleate f - actin in n - cadherin cell contacts , as previously shown for e - cadherin at cell junctions in epithelial cells . since arp3 staining is comparable between tmod1 and tmod1 mature lens fibers , tmod1 does not control arp2/3-driven f - actin branching , which is thus unlikely to play a role in formation or maintenance of the large paddle contours in mature lens fibers . we observe that tmod1 , 2-spectrin , -actinin , fimbrin , and arp3 are localized to small puncta at the base of some but not all protrusions and/or paddles in mature fiber cells . there are several possible reasons why proteins are not consistently localized to all protrusions and/or paddles . first , since the protrusions and paddles are complex 3d structures , confocal imaging of single optical sections ( as presented here ) may miss staining signals from out - of - plane structures . second , our fixation conditions may lead to variable epitope accessibility in the case of proteins that are components of dense f - actin cytoskeletal structures . third , preparation of single fiber cells that dissociates fibers from tightly adhered neighboring cells may lead to small rips and tears along the membrane , leading to a loss in staining at some protrusions and paddles . however , based on staining of single fibers with wheat germ agglutinin ( data not shown ) , plasma membrane continuity does not appear to be compromised by our preparation method . finally , fourth , it is attractive to speculate that constant membrane remodeling occurs in lens fiber cells during differentiation and maturation , requiring dynamic changes in the actin cytoskeleton . this notion is supported by the presence of a pool of monomeric g - actin in the lens , consistent with ongoing f - actin polymerization and depolymerization in fiber cells . actin cytoskeleton reorganization is a hallmark of normal lens fiber differentiation , and there is an increase in polymerized f - actin versus monomeric g - actin in newly formed lens fibers . since our staining experiments capture a single snapshot in time of protein localization , we miss the dynamic changes that may remodel the membrane . in summary , we have developed a novel technique to immunostain single lens fiber cells and have shown that tmod1 stabilizes the spectrin  actin network and -actinin  crosslinked antiparallel f - actin bundles in the valleys between large paddles to maintain their structure . on the other hand , formation of small protrusions is likely facilitated by arp3-nucleated actin networks , as well as fimbrin - crosslinked parallel f - actin bundles , which do not depend on tmod1 . thus , this work reveals specific abps required for the formation of large paddles between lens fibers , and suggests that large paddles between mature fiber cells are needed for lens mechanical integrity .
purposeto elucidate the proteins required for specialized small interlocking protrusions and large paddle domains at lens fiber cell tricellular junctions ( vertices ) , we developed a novel method to immunostain single lens fibers and studied changes in cell morphology due to loss of tropomodulin 1 ( tmod1 ) , an f - actin pointed end  capping protein.methodswe investigated f - actin and f - actin  binding protein localization in interdigitations of tmod1+/+ and tmod1/ single mature lens fibers.resultsf-actinrich small protrusions and large paddles were present along cell vertices of tmod1+/+ mature fibers . in contrast , tmod1/ mature fiber cells lack normal paddle domains , while small protrusions were unaffected . in tmod1+/+ mature fibers , 
 tmod1 , 2-spectrin , and -actinin are localized in large puncta in valleys between paddles ; but in tmod1/ mature fibers , 2-spectrin was dispersed while -actinin was redistributed at the base of small protrusions and rudimentary paddles . 
 fimbrin and arp3 ( actin - related protein 3 ) were located in puncta at the base of small protrusions , while n - cadherin and ezrin outlined the cell membrane in both tmod1+/+ and tmod1/ mature fibers.conclusionsthese results suggest that distinct f - actin organizations are present in small protrusions versus large paddles . 
 formation and/or maintenance of large paddle domains depends on a 2-spectrin  actin network stabilized by tmod1 . 
 -actinin 
 crosslinked f - actin bundles are enhanced in absence of tmod1 , indicating altered cytoskeleton organization . 
 formation of small protrusions is likely facilitated by arp3-branched and fimbrin - bundled f - actin networks , which do not depend on tmod1 . 
 this is the first work to reveal the f - actin  associated proteins required for the formation of paddles between lens fibers .