myosins are a large and diverse family of molecular motors important for cell migration and motility .
the human genome encodes 39 myosin genes , subdivided into 12 different classes ( berg et  al . , 2001 , peckham and knight , 2009 ) .
the remaining three encode the non - muscle ( nm ) myosin isoforms 2a , 2b , and 2c , which contribute to cell shape , adhesion , and cytokinesis ( mogilner and keren , 2009 , vicente - manzanares et  al . , 2009 ) .
myosin isoforms in the remaining classes contribute to a wide range of functions , including organelle trafficking , membrane tethering , golgi organization , actin organization , and actin polymerization ( hartman and spudich , 2012 ) .
early studies have shown that 811 different myosin isoforms are co - expressed in epithelial cell lines , leukocytes , liver cells , and myoblasts ( bement et  al .
some myosin isoforms are expressed widely , whereas others ( e.g. , myo7a and myo3 ) are restricted to a small tissue subset ( dos and burnside , 2000 , hasson et  al . , 1995 ) .
it has never been determined how variation in myosin expression profile between closely related cell types contributes to a variation in cellular phenotype .
modulating myosin expression could help to drive a cell toward a more migratory phenotype and , therefore , metastasis in cancer .
here we determined the myosin isoform expression profile in a range of prostate cell lines and in  silico and then investigated four of the overexpressed myosin isoforms to uncover how each contribute to the more highly metastatic phenotype of pc-3 cells ( pulukuri et  al . , 2005 ) .
we analyzed myosin expression for all 26 of the non - muscle myosin genes in the three most widely used prostate cancer cell lines : pc-3 , du145 , and lncap ( weber et  al . , 2004 ) .
pc-3 cells are considered to have a higher metastatic potential than lncap cells ( aalinkeel et  al . , 2004 ) .
class 2 muscle myosin isoforms were excluded because they are not expressed in non - muscle cells .
we also analyzed a matched pair of normal ( 1535np ) and cancerous ( 1535ct ) cell lines derived from the prostate of the same patient ( bright et  al .
a core of 12 myosin genes were expressed in all cell lines tested , as demonstrated by rt - pcr ( table s1 ) .
however , du145 cells additionally expressed two myosin isoforms , myo7a and myo3 , normally only expressed in the cochlea , retina , testis , lung , and kidney ( hasson et  al . , 1995 ) or in the retina and  pancreas ( dos and burnside , 2000 ) respectively , and , therefore , we did not use these cells in further experiments , although , for completeness , the qpcr analysis on these cells is included ( figure  s1 ) .
expression levels of myo1b , myo1d , myo1e , myo9b , myo10 , and myo18a were significantly higher in pc-3 than in lncap cells by qpcr ( figure  1a ) .
myo1b and myo10 expression levels were also significantly higher in 1535ct than in 1535np cells ( figure  1b ) .
an in  silico analysis ( figure  1c ) showed that myo1b , myo1d , and myo10 levels were significantly higher in metastatic tumors than in benign tissue , suggesting that this trend is also found in  vivo .
myo1e and myo18a expression levels were also higher in 1535ct cells compared with 1535np cells , although this difference was not significant , and the in  silico analysis did not show any significant differences in expression ( figure  1c ) .
myo6 expression levels were significantly lower in pc-3 cells compared with lncap ( figure  1a ) , lower in 1535ct than in 1535np cells ( figure  1b ) , and highest in localized medium - grade tumors ( figure  1c ) , as reported earlier ( dunn et  al . , 2006 ,
myo9b expression levels were increased in tumors compared with benign tissues ( figure  1c ) .
levels of myh9 , the only non - muscle myosin 2 gene we found to be expressed in prostate cancer cells , did not change at the mrna level ( figure  1a ) between lncap and pc-3 cells or between normal , tumor , or metastatic samples in the in  silico analysis .
western blotting for myo1b , nm2a , myo6 , myo9b , myo10 , myo18a , and nm2a in pc-3 and lncap cells ( figures 2a and 2b ) showed similar trends in protein expression levels .
in pc-3 cells , high endogenous levels of myo10 were associated with a high number of filopodia ( figures 2c and 2d ) , in which myo10 was localized to the tips ( figure  2f ) , as expected from its known role in filopodium formation ( berg and cheney , 2002 , berg et  al . , 2000 ) .
in contrast , both filopodium number and myo10 expression levels were low in lncap cells ( figure  2c ) , staining was diffuse , and myo10 was often absent from filopodial tips ( figures 2e and 2f ) .
upregulation of myo10 in breast cancer cells has been linked to expression of mutant p53 ( arjonen et  al . , 2014 ) . however , lncap cells are p53 wild - type , and pc-3 cells are p53-null ( carroll et  al . , 1993 ) , suggesting that , in this case , there is no link between myo10 overexpression and expression of mutant p53 . in du145 cells , which do express mutant p53 ,
myo10 expression is slightly higher , and numbers of filopodia are increased compared with lncap cells ( figures s1a and s1b ) , but both are lower compared with pc-3 cells .
myo1b localized to organelles in both cell types ( figures 2d and 2e ) , as expected from its roles in trafficking of endosomes , multivesicular bodies , and lysosomes ( cordonnier et  al . , 2001 ,
higher myo1b expression in pc-3 cells was associated with an additional localization of myo1b to actin - rich structures at the plasma membrane and filopodia ( figure  2d ) , consistent with an earlier study ( tang and ostap , 2001 ) .
myo9b and myo18a were both enriched in membrane ruffles / lamellipodia in pc-3 cells ( figure  2d ) , consistent with myo18a s role in modifying actin organization in the lamellipodium ( hsu et  al . , 2010 ) and
myo9b s role in cell polarity and recruitment of rhogap to the lamellipodium ( hanley et  al .
the higher endogenous expression levels of myo1b , myo9b , and myo10 in more metastatic cell types / tissue suggested that they all contribute to the cellular phenotype of metastatic cells .
we therefore used sirna knockdown ( kd ) to determine the effects of reducing their expression levels in pc-3 cells on cell morphology and cell migration .
we also investigated myo18a because the interaction of myo18a regulates nm2a filaments ( billington et  al . , 2015 ) and , therefore , may also influence cell migration and phenotype .
sirna - mediated kd for 72  hr significantly reduced expression levels of each myosin isoform in pc-3 cells ( figure  3a ) and altered their morphology ( figure  3b ) .
the spread area of the cells increased up to 3-fold ( figure  3c ) .
kd of myo10 , but not myo1b , myo9b , or myo18a , also significantly reduced the numbers of filopodia ( figure  3d ) . although the increase in cell area in myo10 kd cells could be explained by the reduction in filopodia , as reported for cos-7 and hela cells ( bohil et  al . , 2006 )
, it does not explain the increased cell area for myo1b , myo9b , and myo18a kd cells , where filopodia are still present .
knockdown of myo9b and myo10 both significantly reduced cell speed 2-fold in a 2d random migration assay ( figures 3e and 3f ) .
in contrast , knockdown of myo1b and myo18a did not affect speed in 2d random migration assays ( figures 3e and 3f ) .
knockdown of myo18a significantly reduced directional persistence in 2d ( figures 3e and 3 g ) , indicating that these cells are less able to polarize .
however , cell migration was inhibited for each myosin in a circular invasion assay ( figure  3h ) that closely mimics 3d invasion ( yu and machesky , 2012 ) . staining for f - actin in circular migration assays ( figure  3i ) showed an increase in actin stress fibers for cells at the border for each myosin knockdown compared with controls .
we also observed distinct changes in the acto - myosin organization following kd of each myosin .
control pc-3 cells ( figures 4a and 4b ) contained few f - actin stress fibers , and nm2a staining was mostly localized to  the lamellae . a  marked increase in centripetal f - actin fibers running parallel to the plasma membrane in the lamella associated with nm2a filaments was characteristic of myo18a kd ( figures 4a and 4b ) .
the appearance of sparse , long stress fibers , associated with nm2a and extended along the length of the cells , was characteristic of myo1b kd ( figures 4a4c ) .
a line profile analysis of the frequency of actin bundles in the lamellae of kd cells showed that the frequency of bundles was reduced significantly ( 2.1  0.1 bundles/m , mean  sem , n  = 9 ) compared with controls ( 2.6  0.2 bundles/m , mean  sem , n  = 9 , p  < 0.5% ) ( figures 4a and 4b ) , suggesting that the actin cytoskeleton is being re - organized .
myo9b kd cells contained a distinctive actin - rich area at the cell periphery from which nm2a was largely absent , in addition to an increase in stress fibers ( figures 4a and 4b ) .
myo10 kd cells showed loss of filopodia and the appearance of distinctive actin bundles in the central region of the cell ( figures 4a and 4b ) .
changes to the f - actin organization were associated with formation of focal adhesions at the edges of the cells , consistent with a more spread cell phenotype ( figure  4c ) .
knockdown of myo1b , myo10 , or myo18a did not change phosphorylation levels of myosin light chain ( mlc ) ( figure  4d ) , suggesting that  nm2a is re - organized rather than activated as a result of their knockdown .
kd did increase mlc phosphorylation 2-fold in cells , and this is likely to contribute to the actomyosin re - organization observed ( figure  4d ) .
these data show that myo10 , myo9b , and myo1b are overexpressed in more highly metastatic cell lines and in metastatic tissue .
high levels of myo10 in pc-3 cells are linked to high numbers of filopodia , and high levels of myo9b are linked to low levels of stress fibers .
both isoforms contribute to a more migratory phenotype , as shown by immunostaining , cell migration , and kd experiments . myo1b and myo18a influence cell morphology and actin organization but have little effect on  migration in 2d , whereas all four isoforms inhibit cell migration in invasion assays .
therefore , changes in expression of several myosin isoforms may contribute to metastasis in prostate cancer .
our finding that myo10-dependent filopodia are likely to be important in prostate cancer agrees with recent similar findings for breast cancer metastasis ( arjonen et  al .
, 2014 , cao et  al . , 2014 ) and non - small lung cell cancer ( sun et  al . , 2015 ) .
filopodia are important but not absolutely required for cell migration because myo10 kd cells can migrate in 2d but with a reduced speed , and other cells lacking filopodia can migrate ( lundquist , 2009 ) .
the increased cell area resulting from myo10 kd agrees with previous findings ( bohil et  al . ,
the central actin bundles in myo10 knockdown cells are reminiscent of actin bundles in filopodia .
fascin is also required for filopodial formation ( vignjevic et  al . , 2006 ) , its overexpression results in multiple filopodia ( vignjevic et  al . , 2006 ) , and fascin levels are also upregulated in prostate cancer ( darnel et  al . , 2009 ) .
myo10 kd may lead to actin bundling in the cell body by excess ( non - phosphorylated ) fascin .
the role of two filopodial proteins , myo10 and fascin , in prostate ( and other ) cancers suggest that filopodium formation is key for metastasis .
myo10 has also been implicated in integrin - mediated adhesion , and any reduction in adhesion resulting from its kd could disrupt signaling to the actin cytoskeleton and , therefore , indirectly result in changes in actin organization .
high levels of myo9b expression in pc-3 cells are likely to contribute to their lack of stress fibers and , therefore , to enhanced migration .
the rhogtpase - activating domain in myo9b inhibits rho , reducing the downstream activity of rock ( rhokinase ) , thereby increasing mlc phosphatase activity , reducing mlc phosphorylation ( reinhard et  al . , 1995 , wirth et  al . , 1996 ) , and , therefore , reducing actin stress fiber formation .
knockdown of myo9b is therefore expected to increase mlc phosphorylation and stress fiber formation , as we observed . in agreement with our findings
, a previous report has shown that cell migration was reduced and mlc phosphorylation increased in macrophages isolated from myo9b knockout mice ( hanley et  al . , 2010 ) .
myo9b has also been implicated in an increased risk of esophageal cancer ( menke et  al . ,
the high levels of myo1b in pc-3 cells and effects of knockdown on 3d invasion , cell shape , and morphology suggest that it , too , has a role in prostate cancer .
myo1b has also been implicated in non - small - cell lung cancers ( ohmura et  al . ,
myo1b ( myr1/mm1 ; gillespie et  al . , 2001 ) regulates actin assembly in vesicular transport ( post - golgi carriers [ almeida et  al . ,
raposo et  al . , 1999 ] ) , and it maintains cortical tension at the plasma membrane , where it specifically associates with dynamic , non - tropomyosin - containing actin filaments ( coluccio and geeves , 1999 , tang and ostap , 2001 )
. high endogenous levels of myo1b in more highly metastatic cells might therefore increase cortical tension , allowing cells to move through stiff extracellular matrices in  vivo , perhaps explaining why knockdown of myo1b only affects migration in 3d but not 2d .
the re - organization of actin and nm2a in myo18a kd cells may arise from its interaction with non - muscle myosin 2 ( nm2 ) .
nm2 forms short filaments ( 300  nm long ) containing 20 molecules ( billington et  al . ,
the assembly / disassembly of non - muscle myosin 2 filaments is dynamic ( shutova et  al . , 2014 ) and
regulated by many different pathways ( vicente - manzanares et  al . , 2009 ) .
myo18a and nm2a can form mixed bipolar filaments in  vitro that are smaller that pure nm2a filaments ( billington et  al . , 2015 ) .
the re - organization of nm2a we observed after knocking down myo18a , without a change to levels of light chain phosphorylation , supports the idea that an interaction between myo18a and nm2a modulates nm2a filament formation and organization in pc-3 cells .
therefore , myo1b , myo9b , myo10 , and myo18a each contribute to the morphology and migration of more highly metastatic pc-3 cells , with each myosin having a specific effect on actin organization .
misregulation of their expression in cells with metastatic potential may allow them to work in concert to generate a torpedo - shaped cell with multiple protrusions that is better able to migrate through a 3d matrix and , therefore , more able to metastasize .
many different drugs have now been developed that can inhibit specific myosin isoforms , including those in classes 1 , 2 , 5 , and 6 ( bond et  al . , 2013 ) .
importantly , these results emphasize that myosin not only uses actin as tracks to walk along but that it is able to actively drive actin organization in cells .
lncap , du145 , and pc-3 cells ( from the atcc ) were grown in rpmi 1640 medium with glutamax ( gibco , life technologies ) supplemented with 10% heat - inactivated fetal bovine serum ( fbs ) and penicillin - streptomycin . 1535np and ct cells ( bright et  al . , 1997 )
they were grown in keratinocyte medium ( gibco , life technologies ) supplemented with 10% heat - inactivated fbs , 1% l - glutamate , antibiotics , bovine pituitary extract , and epidermal growth factor .
the antibodies used were as follows : myo6 ( h-215 , santa cruz biotechnology ) ; myo10 ( hpa024223 , sigma ) ; total erk ( p44/42 mitogen - activated protein kinase [ mapk ] , cell signaling technology ) ; myo18a , a gift from prof .
( chang gung university , taiwan ; hsu et  al . , 2010 ) or from genscript ; nm2a ( prb-440p , covance ) ; paxillin ( sab4502553 , sigma ) ; myo1b ( hpa013607 , sigma ) ; phospho - myosin light chain ( cell signal ) ; myo9b ( proteintech ) ; and glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) ( abcam ) .
hrp - conjugated secondary antibodies and fluorescent phalloidin were from sigma , and fluorescent secondary antibodies were from molecular probes .
sigenome smartpool sirna ( ge healthcare , dharmacon ) was used to silence myosins in pc-3 cells .
cells were seeded at a density of 20,000 cells / ml in growth media and allowed to adhere and grow overnight .
maximum kd was achieved after 72  hr . the rneasy mini kit ( qiagen )
rt - pcr was used to detect which myosin isoforms were expressed ( table s1 ) . real - time pcr using sybr green was used to investigate the expression levels of expressed myosins ( see table s2 for primer sequences ) .
cells were lysed ( 30  min , 4c ) in lysis buffer ( 150  mm nacl , 0.05  m tris [ ph 8 ] , 1% triton x-100 , and 1  mm edta [ ph 8 ] with protease inhibitor cocktail ( thermo scientific ) .
lysates were clarified by centrifugation , protein content was quantified by bicinchoninic acid ( bca ) assay , and then samples were mixed with 2 laemmli buffer for use in protein gels ( 4%20% or 7.5% ) and blots .
chemiluminescence detection ( supersignal west pico , thermo scientific ) used multiple exposures to ensure signal linearity . if required , membranes were stripped using restore western blot stripping buffer ( thermo scientific ) and re - probed .
cells were grown on glass coverslips , fixed with 2% paraformaldehyde in pbs , and stained using standard procedures ( swailes et  al . , 2006 ) .
, cells were plated onto a glass - bottomed 96-well plates , transfected with non - targeting sirna or with myosin kd sirna ( three replicates each ) , serum - starved 48  hr later for 24  hr , and then treated with hepatocyte growth factor ( hgf ) ( 25  ng / ml ) for filming .
a minimum of three fields from each replicate was selected for imaging , over 14  hr at 5-min intervals using differential interference contrast ( dic ) optics , and a 20 lens ( 512   512 total pixel size , 2   2 binning ) on a deltavision system .
cell migration was analyzed using imagej software ( mtrackj plugin ) . to perform the 3d - like circular invasion assay ( yu and machesky , 2012 ) ,
cell - free space was created using cell stoppers ( ibidi ) . after removing the stopper
, cells were covered with a thin layer of matrigel ( 4  mg / ml ) and normal medium and allowed to grow and migrate for another 2448  hr .
data are presented as mean  sd for at least three separate experiments ( n  3 ) . a two - way anova was used to compare differences between groups , and statistical significance was accepted for p  0.05 .
 summarywe investigated the myosin expression profile in prostate cancer cell lines and found that myo1b , myo9b , myo10 , and myo18a were expressed at higher levels in cells with high metastatic potential . 
 moreover , myo1b and myo10 were expressed at higher levels in metastatic tumors . using an sirna - based approach 
 , we found that knockdown of each myosin resulted in distinct phenotypes . myo10 knockdown ablated filopodia and decreased 2d migration speed . 
 myo18a knockdown increased circumferential non - muscle myosin 2a - associated actin filament arrays in the lamella and reduced directional persistence of 2d migration . 
 myo9b knockdown increased stress fiber formation , decreased 2d migration speed , and increased directional persistence . 
 conversely , myo1b knockdown increased numbers of stress fibers but did not affect 2d migration . in all cases , 
 the cell spread area was increased and 3d migration potential was decreased . 
 therefore , myosins not only act as molecular motors but also directly influence actin organization and cell morphology , which can contribute to the metastatic phenotype . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions