the uptake of large amounts of lipids from plasma lipoproteins via endocytosis by macrophages in the artery wall to form lipid - engorged foam cells is an important initiating event in the development of atherosclerosis , a major cause of heart disease .
the main lipoprotein implicated is low - density lipoprotein ( ldl ) , but oxidative modification of the particles , a process which can occur within the vessel wall , is required before foam cell formation is induced .
the myosin superfamily is a group of actin - associated motor proteins that use atp hydrolysis to generate force or directional movement along actin filaments [ 2 , 3 ] .
many new classes of myosin have been identified since the discovery , over seventy years ago , of myosin ii (  conventional  myosin ) , which produces contractile force in muscle cells ; to date , 35 classes of myosin proteins have been characterised in mammalian cells .
it is now known that these various classes of myosin motor are involved in a diverse range of intracellular processes , ranging from cell migration and division to cell anchorage and transport of cargo proteins along actin filaments .
myosin vi ( myo6 ) , a member of the  unconventional  myosin classes , is an unusual member of the myosin superfamily in that its movement along actin filaments is towards the pointed (  ) end of an actin filament , the opposite direction to myosins of other classes .
for this reason , myo6 is ideally suited for a role in endocytosis , which involves vesicle formation and/or transport away from the plasma membrane .
evidence suggests that it may play a part in organization and/or anchorage of endocytic machinery , in the provision of force required for invagination and/or vesicle fission , and during transport of vesicles through the cell cortex [ 68 ] .
attachment to vesicles is achieved through direct interaction with adaptor proteins ( aps ) , including disabled 2 ( dab2 ) ( which binds directly to receptors or indirectly via adaptor protein-2 ( ap2 ) ) and gaip interacting protein c terminus 1 ( gipc1 ) [ 79 ] .
myo6 can be alternatively spliced in two regions of its cargo binding domain , giving rise to either a small insert ( 9aa ) ( si ) and/or a large insert ( up to 32aa ) ( li ) .
thus , four variants are possible : myo6(+si , + li ) , myo6(si , + li ) , myo6(+si , li ) , and myo6 without inserts ( myo6(i ) ) . these variants
are differentially expressed and appear to have distinct cellular targets and functions [ 6 , 7 ] . although the inserts contain no functional motifs or binding sites , they may affect the structure and therefore the affinity of myo6 for its binding partners [ 9 , 11 ] .
endocytosis may occur by phagocytosis , the uptake of large particles , or by pinocytosis , the internalization of fluid and small particles [ 10 , 12 ] .
phagocytosis is a regulated and selective process requiring receptor - mediated target recognition , but pinocytosis can be nonselective ( macropinocytosis ) or can be receptor - mediated .
clathrin - mediated endocytosis ( cme ) is the most studied form of receptor - mediated pinocytosis , but other forms include caveolin - mediated endocytosis , as well as clathrin - and - caveolin - independent endocytosis [ 10 , 12 ] .
most evidence supporting a role for myo6 in endocytosis relates to cme : it has been shown to colocalise with clathrin , dab2 , and ap-2 in polarised cells [ 1315 ] and with uncoated vesicles and gipc1 in unpolarised cells [ 13 , 16 ] .
in addition to cme , myo6 has been found localized to sites of macropinocytosis in fibroblasts , and holt et al .  
have reported that loss of myo6 function in bone marrow - derived murine dendritic cells causes enhancement of macropinocytotic uptake , but its exact function in this process remains unclear [ 7 , 18 ] . given the importance of endocytosis in the uptake of lipoproteins by macrophages , it is possible that myo6 may have a role in foam cell formation and thus play a part in atherogenesis .
little is known , however , about the expression and function of myo6 and its binding partners in macrophages .   expression of myo6 , dab2 , and ap-2   has been detected in murine macrophages and expression of gipc in human peripheral blood monocytes , but , except for one report which found myo6 mrna in macrophages derived from the human monocyte cell line , thp-1 , no information is available on expression of these proteins in human macrophages .
moreover , as far as we are aware , splice variant expression , subcellular localisation , and protein - protein interactions of myo6 have not been studied previously in any macrophage type .
the aims of this study were to investigate the expression and function of myo6 and related proteins in human macrophages and to test the hypothesis that myo6 plays a role in the endocytosis of ldl and/or oxldl by these cells .
expression of myo6 ( including splice variants ) , dab2 , ap-22 ( one of the large subunits of the ap-2 heterotetrameric complex ) , and gipc1 was demonstrated in both thp-1 macrophages and primary human monocyte - derived macrophages ( hmdm ) , and their subcellular location and interactions within the cells were investigated . to study the possible role of these proteins in macrophage endocytosis
, thp-1 macrophages were used to evaluate the effects of ldl and oxldl on the expression of mrna and protein for myo6 , dab2 , ap-2 , and gipc1 , as well as the effects of inhibition of myo6 and dab2 expression by small interfering rna ( sirna ) on the uptake of the lipoproteins by the cells .
the thp-1 cell line was established 30 years ago , and since then it has been used very extensively to study the role of monocyte / macrophages in cardiovascular disease .
a recent comprehensive review of the value of this model   concluded that , in defined conditions , thp-1 cells resemble hmdm and mimic the changes seen in atherosclerosis and that , provided results are interpreted cautiously , they are suitable for the study of the mechanisms by which these cells affect vascular function .
the present work shows that the expression and subcellular location of myo6 and related proteins are generally similar in hmdm and thp-1 macrophages , but because of the practical difficulties in obtaining large numbers of hmdm , it was not possible to use primary cells in all of our experimental approaches .
fetal bovine serum and penicillin / streptomycin were obtained from gibco ( paisley , uk ) , and dmem and l - glutamine were from paa uk ( yeovil , uk ) .
rpmi 1640 medium , -mercaptoethanol , 4-phorbol 12 myristate 13-acetate ( pma ) , oil red o , ethidium bromide , and fluorescein - isothiocyanate- ( fitc- ) conjugated goat anti - mouse igg were supplied by sigma aldrich ( poole , uk ) .
4,6-diamidino-2-phenylindole ( dapi ) 1,1-dioctadecyl-3,3,33-tetramethylindo - carbocyanine perchlorate ( dii ) , rhodamine conjugated phalloidin , alexa488 conjugated goat anti - rabbit- , and alexa555 donkey anti - rabbit igg were purchased from invitrogen molecular probes ( paisley , uk ) .
myo6 ( h-215 ) and dab2 ( h-110 ) rabbit polyclonal igg and normal donkey serum were from santa cruz biotechnology inc .
other antibodies including mouse polyclonal anti - ap-22 and rabbit polyclonal anti--microglobulin igg ( abcam , cambridge , uk ) , mouse polyclonal anti - gipc1 igg ( abnova , heidelberg , germany ) , rabbit polyclonal anti--actin ( cell signaling technology , hitchin , uk ) , and horse radish peroxidase - conjugated goat anti - mouse and goat anti - rabbit igg ( thermo fisher scientific , cramlington , uk ) were from various suppliers as indicated , and normal goat serum was from dako uk ltd ( ely , uk ) .
thp-1 monocytes were cultured in suspension in rpmi-1640 media , supplemented with 10% heat inactivated ( 56c , 30  min ) fetal bovine serum ( fbs ) , 1% penicillin / streptomycin , and 0.1%  -mercaptoethanol ( culture medium ) .
for differentiation into macrophages , monocytes were incubated with 200  ng / ml phorbol 12-myristate 13-acetate ( pma ) ( 200  ng / ml ) for 72  h. prior to experimental treatment , the medium containing pma and nonadherent or dead cells was removed , the cells were washed twice , and fresh culture medium was added .
cells were incubated with or without treatment for up to 5 days , with media changes every 48  h. for hmdm preparation , primary human monocytes were isolated from the blood of healthy adult volunteers with ethical approval from the east london research ethics committee .
blood was collected into tubes containing 15% edta ( v  :  v ) and processed immediately by the addition of pbs ( 1  :  1 , v  :  v ) , layering over 15  ml lymphoprep ( axis healthcare ltd , borehamwood , herts , uk ) and centrifugation at 800  g ( 30  min , 20c ) .
the mononuclear cell layer was collected , mixed with an equal volume of ice - cold pbs containing 0.4% ( v / w ) trisodium citrate , and centrifuged at 800  g ( 5  min , 4c ) .
after removal of the supernatant , the cell pellet was re - suspended in 0.2% ( w  :  v ) nacl for 30  sec at 4c to lyse any remaining red blood cells and 1.6% ( w  :  v ) , nacl was added , and the tubes were centrifuged as previously mentioned .
resuspension in pbs containing 0.4% ( w  :  v ) trisodium citrate and centrifugation were then repeated 6 to remove contaminating platelets .
the final pellet was resuspended in rpmi-1640 medium containing 5% fbs and 1% penicillin / streptomycin , and the cells were incubated at 37c in 5% co2 for 7 days to allow differentiation into macrophages .
examination by light microscopy indicated that the cells showed a macrophage phenotype after 6 days ; cells were used on the 7th day after washing with pbs ( 4 ) to remove any remaining nonadherent cells .
cos-7 adherent cells were cultured in dmem maintenance media supplemented with 10% fbs , 1% penicillin / streptomycin , and 1% l - glutamine .
total rna was extracted from hmdm , thp-1 macrophages , and cos-7 cells using an extraction kit ( sigma - aldrich , poole , uk ) and rnase - free dnase ( qiagen , crawley , uk ) as recommended by the manufacturers . for reverse transcription , rna
( 1  g ) was used to generate cdna using omniscript reverse transcriptase ( qiagen ) and oligo-(dt ) primers according to the manufacturers ' instructions . for expression studies in untreated cells ,
pcr was used to amplify gene products for myo6 , the small and large insert regions of myo6 , dab2 , ap-22 , gipc1 , and in geneamp pcr system 9700 ( applied biosystems ) using the primers shown in table 1 and the following protocol : denaturing at 95c ( 5  min ) , followed by 35 cycles consisting of 30  sec at 94c , 90  sec at the appropriate specific annealing temperatures and extension at 72c for 60  sec , with a final extension at 72c for 10  min .
pcr products were visualised on 1.2% or 3% agarose gels containing 0.01% ethidium bromide ( v  :  v ) .
gels were viewed and photographed under uv light using the chemidoc xrs scanner ( bio - rad , hemel hempstead , uk ) . to determine the effects of ldl or oxldl on mrna expression
, thp-1 macrophages were incubated with the lipoproteins ( 50  g protein / ml ) for 8 or 24  h , and total rna was then extracted and reverse transcribed as described previously . the abundance of mrna transcripts for myo6 , dab2 , ap-22 , gipc1 , -actin , 2-macroglobulin , and ribosomal protein l13a was assessed by quantitative pcr ( qpcr ) in an opticon 2 lightcycler system ( mj research , waltham , massachusetts , usa ) using the primers shown in table 1 employing the following protocol : denaturing at 94c ( 2  min ) , followed by 37 cycles of amplification consisting of denaturation at 94c ( 15  sec ) , incubation at an appropriate , specific annealing temperature ( 1  min ) , followed by extension at 72c ( 1  min ) .
threshold cycle values were determined using opticon monitor 3 software and quantified using the standard curve for each gene .
normalisation factors ( nf ) were calculated from the geometric means of the three most stably expressed reference genes ( -actin , 2-macroglobulin , and ribosomal l13a protein ) as determined using genorm software or , for sirna studies , to the reference gene 2-microglobulin run on the same qpcr plate with its own standard curve . in the latter case ,
annealing temperatures ( table 1 ) were adjusted to a temperature suitable for both the genes of interest and 2-microglobulin . for analysis of protein expression by western blotting
, cells were washed twice with pbs and treated with ripa lysis buffer ( 150  mm nacl , 50  mm tris ( ph 8.0 ) , 1% triton x-100 , 0.5% deoxycholate ( doc ) , 0.1% sds ) containing protease inhibitor cocktail ( 10  l / ml ) ( 5  min , 4c ) .
lysates were agitated for 30  min at 4c , centrifuged at 93  g ( 10  min at 4c ) to remove cell debris , and then denatured and reduced in nupage lds sample buffer ( 2  :  1 , v  :  v ) containing 10%  -mercaptoethanol solution at 70c for 10  min . proteins ( 1030  g ) were separated by sds - polyacrylamide electrophoresis ( 8% or 10% resolving gel with 5% stacking gel or precast mini - protean tgx biorad graded gels ( 415% ) ( biorad ) ) and transferred to polyvinylidene fluoride ( pdvf ) membranes .
blocking was carried out in 5% ( w  :  v ) nonfat milk powder at room temperature for 1  h , and membranes were then incubated with polyclonal rabbit anti - myo6 ( 1  :  200 ) , anti - dab2 ( 1  :  400 ) , anti--actin ( 1  :  1,000 ) or anti--microglobulin ( 1  :  700 ) , or mouse anti - gipc1 ( 1  :  500 ) igg overnight at 4c with agitation . after washing in tbst ( 4  15  min ) , horseradish peroxidase - conjugated goat anti - rabbit or anti - mouse secondary antibodies ( 2-microglobulin , dab2 , myo6 , 1  :  8,000 ; -actin , gipc1 , 1  :  10,000 ) were added and incubations were continued for 1  h at room temperature with agitation .
finally membranes were washed in tbst ( 4  15  min ) and bands were visualized using enhanced chemiluminescence ( ge healthcare , little chalfont , uk ) .
band densities were digitized from film to 12  bit images using a chemidoc xrs ( bio - rad ) and quantified as mean grey values measured over identical areas using quantity one software ( bio - rad ) .
cls fluorescence microscopy was conducted using either a leica sp5 or a zeiss lsm510 confocal microscope .
for all experiments , thp - macrophages or hmdm were incubated in 24-well plates containing coverslips , which were removed for mounting at the end of the procedure .
coverslips were washed twice in 1  pbs , fixed with 4% formalin ( v  :  v ) for 15  min , and followed by cell permeabilisation in 0.25% triton x-100 for 5  min prior to blocking ( 1  h , 3% ( w  :  v ) bsa ) with normal goat ( ngs ) or donkey ( nds ) serum ( 1% w  :  v ) .
igg polyclonal rabbit anti - myo6 , -gipc1 ( 1  :  100 , v  :  v ) , -dab2 ( 1  :  200 , v  :  v ) , or polyclonal mouse anti - ap-22 ( 1  :  100 , v  :  v ) ( diluted in 0.1% ( w  :  v ) bsa and 1% ngs / nds in pbs ) was then added . after 1  h ,
cells were washed ( 0.5% bsa in pbs , 5  min  4 and 15  min  1 ) and incubated with igg polyclonal secondary antibodies conjugated to a specific fluorochrome as follows : goat anti - rabbit antibody conjugated to alexa fluor 488 ( 1  :  1,000 , v  :  v ) , donkey anti - rabbit antibody conjugated to alexa fluor 555 ( 1  :  1,000 , v  :  v ) , or goat anti - mouse antibody conjugated to fluorescein isothiocyanate ( fitc ) ( 1  :  25 , v  :  v ) dilutions in 0.1% bsa and 1% ngs in pbs ( w  :  v ) .
where double immunofluorescence staining was used , compatible pairs of primary or secondary antibodies were incubated with cells simultaneously .
cells were then washed in pbs ( 5  min  3 , then 15  min  1 ) .
to visualise cell nuclei , cells were incubated ( 3  min ) with 300  nm 4,6-diamidino-2-phenylindole ( dapi ) , a nucleic acid stain .
the structure of f - actin was preserved prior to visualisation , with minimal loss of free myosin , by incubating cells ( 45  sec ) with buffer containing nacl , 137  mm ; kci , 5  mm ; na2hpo4 , 1.1  mm ; kh2po4 , 0.4  mm ; nahco3 , 4  mm ; glucose , 5.5  mm ; mgcl2 , 2  mm ; egta , 2  mm ; pipes , 5  mm ; ph 6.0 - 6.1 [ 25 , 26 ] supplemented with 0.32  m sucrose , 0.1% triton x-100 , and 1  g / ml phalloidin .
cells were immediately incubated with 165  nm phalloidin conjugated to rhodamine , as a fluorescent marker ( 20  min ) .
areas of fluorescence signal were detected by application of a threshold and the mean fluorescence intensity quantified using volocity ( 5.4 ) 3d image analysis software ( perkinelmer , waltham , ma , usa ) .
g / ml ) was isolated from human plasma obtained from the national blood service ( north london , uk ) , using sequential density gradient ultracentrifugation .
final preparations of native ldl ( nldl ) were dialysed against 0.9% nacl ( w / v ) containing 10  m edta ( 5l  4 ) . for ldl used in oxidation studies ,
human plasma was used within 1 month of purchase and ldl within 2 weeks of isolation .  
ldl was oxidised by incubation with cuso4 ( 5  m ) for 6  h , oxidation was terminated by the addition of edta ( 1% , 50  l / ml v / v ) , and the extent of oxidation was assessed by measuring the concentration of malondialdehyde ( mda ) ( nmol / mg ldl protein ) using the thiobarbituric acid reactive substances ( tbars ) assay .
cuso4 was removed by dialysis at 4c against 0.9% nacl ( w / v ) containing 10  m edta ( 5l  4 ) .
for labelling of nldl and oxldl with the fluorescent probe 1,1-dioctadecyl-3,3,3,3-tetramethylindo - carbocyanine perchlorate ( dii ) , lipoprotein deficient serum ( lpds ) , prepared from human plasma by sequential density gradient ultracentrifugation , was added to the ldl preparations ( 1  mg ldl protein/2  ml lpds ) followed by dii ( 300  g dii / mg ldl protein ) . to prevent oxidation , edta
was added to the preparations at a final concentration of 10m , and the mixture was incubated for 8  h at 37c with shaking .
the dii labelled lipoproteins ( dii - nldl / oxldl ) were then isolated by ultracentrifugation at d 1.063  g / ml ( 96,919  g , 15  h , 4c ) and dialysed as described previously before use .
small interfering rna ( sirna ) sequences designed to target myo6 ( combination of two sequences ) , dab2 , ap-22 ( table 2 ) , or gipc1 ( sequence not released by the manufacturer ) were used to inhibit the expression of their cognate genes .
thp-1 macrophages ( 6  10 well ) were transfected with sirna using hiperfect transfection reagent ( qiagen , crawley , uk )
. 5  nm ( ap-22 , gipc1 , dab2 ) or 40  nm ( myo6 ) sirna was added to 100  l ripa-1640 medium , then 20  l hiperfect was added , and the mixture was incubated for 10  min at room temperature to allow sirna - hiperfect complexes to form prior to their addition ( 100  l ) to cells . in all experiments ,
a nonsilencing scrambled sirna ( allstars negative control , qiagen ) , which has no known homology to any mammalian genes , was used as a control . to assess the effects of inhibition of gene expression on the uptake of nldl and oxldl by thp-1 macrophages
, lipoproteins were labelled with the fluorescent probe dii and uptake was assessed using fluorescence wide - field microscopy .
cells were seeded in 24-well plates ( 1.6  10 cells / well ) and incubated with sirna for 4872  h. dii - labelled ldl or oxldl ( 50  g protein / ml ) was then added , and the incubations , continued for periods up to 24  h. cells were fixed and imaged using a leica inverted dmrib wide - field microscope .
protein concentrations of cell lysates were determined by the method of lowry   and those of nldl and oxldl were obtained using peterson 's modification of lowry 's method .
the total cholesterol content of lipoproteins was determined by enzymatic analyses using commercially available kits ( thermo fisher scientific , cramlington , uk ) . statistical analysis was performed using student 's t test or one- or two - way anova ( followed by bonferroni 's multiple comparison test ) , as indicated in the text .
to determine whether myo6 and its binding partners are expressed in macrophages , total rna was isolated from untreated thp-1 macrophages and hmdm and then used to generate cdna for amplification of gene products by conventional pcr .
bands at the expected product size ( table 1 ) were detected for myo6 ( figure 1(a ) ) and also for dab2 ( figure 1(b ) ) , the large subunit of ap-2 , ap-22 , which was used to detect ap-2 expression ( figure 1(c ) ) and gipc1 ( figure 1(d ) ) . in all cases , no bands were found for any of the genes when pcr was carried out in the absence of either primers ( data not shown ) or cdna .
these results demonstrate that both thp-1 macrophages and hmdm express mrna encoding myo6 , dab2 , ap-22 , and gipc1 .
although previous studies in mammalian cells have established that myo6 can be expressed as four variants due to alternative splicing of the li and si regions of the tail domain [ 6 , 11 ] , it is not known which splice variants are expressed in macrophages .
dance et al .   demonstrated the presence of myo6 both with and without the li and si in cos-7 cells , and we have used the same primer sequences to investigate their expression in thp-1 macrophages and hmdm .
we found bands identical to those reported by dance et al .   for myo6 variants in cos-7 cells ( figure 2 ) , and these were used as markers of band identity .
figure 2 shows that bands corresponding to myo6(li ) ( figure 2(a ) ) and myo6(si ) ( figure 2(b ) ) in cos-7 cells were also found in thp-1 macrophages ( figures 2(a ) and 2(b ) , white arrows ) . a band of product size equivalent to myo6(+si ) mrna ( figure 2(b ) , green arrows )
was also detected in thp-1 cells , although at a lower level relative to myo6(si ) in comparison to cos-7 cells .
expression of myo6(+li ) was low in cos-7 cells , but a band corresponding to this variant was clearly present in thp-1 macrophages , and these cells also expressed number of other transcripts of larger size ( figure 2(a ) , blue arrows ) .
hmdm showed similar patterns of expression of myo6 splice variants to those observed in thp-1 cells , with myo6(li ) and multiple bands of myo6(+li ) ( figure 2(c ) ) and myo6(si ) ( figure 2(d ) ) present ; however , myo6(+si ) was not detected in these cells ( figure 2(d ) ) .
immunoblotting indicated the presence of a protein of the molecular weight of dab2 ( 96/67  kda ) in 3 lysates from thp-1 macrophages and in cells from 3 individual donors for hmdm .
in addition , a band corresponding to the molecular weight of myo6 was clearly present in 3 lysates of thp-1 cells ( one example is shown in figure 3(b ) ) . for hmdm ,
a weak band corresponding to myo6 was visible in cells from 2 donors when 45  g protein was loaded ( one example is shown in figure 3(b ) ) but not when a lower amount of protein ( 35  g ) was used ( data not shown ) .
the expression of myo6 in hmdm , however , was clear in the immunofluorescence experiments described later .
in addition , ap-22 protein could not be detected by immunoblotting , although its presence in thp-1 macrophages was demonstrated by immunofluorescence ( see later ) .
immunofluorescence was used to explore the distribution and interactions of myo6 and associated proteins in thp-1 macrophages and hmdm .
the proteins were detected using alexa488- ( myo6 , dab2 ) or fitc-(ap-22 ) conjugated secondary antibodies ( green ) and where appropriate , dapi ( blue ) and rhodamine - conjugated phalloidin ( red ) were employed as counterstains for the nucleus and f - actin , respectively .
no green fluorescence was observed in negative controls when cells were incubated with fitc- or alexa488-conjugated secondary ab in the absence of the primary antibodies .
ap-22 and dab2 were located mainly around the cell periphery in a punctate staining pattern in both thp-1 macrophages ( figures 4(a ) and 4(c ) ) and hmdm ( figures 4(b ) and 4(d ) ) . by contrast
, myo6 was found to exhibit a more diffuse staining in both cell types ( figure 5 ) and was found within membrane protrusions and membrane ruffles along the leading edge of the cells ( thp-1 cells figures 5(a ) , and 5(b ) , hmdm , figures 5(c ) and 5(d ) ) .
association between myo6 and f - actin was studied using fluorescence labelling techniques in both thp-1 macrophages and hmdm ( figure 6 ) .
confocal images showed that , as expected , myo6 appeared to be associated with f - actin , with similar distributions mainly found around the cell periphery in both types of macrophages ( figures 6(a)(ii ) and 6(b ) ) , including possible podosome attachment sites in thp-1 cells ( figure 6(a)(i ) ) . to assess the extent of interaction between myo6 and ap-22 , double immunofluorescence experiments were carried out in thp-1 macrophages in which myo6 was labelled with alexa555-linked secondary antibody to give red fluorescence ( figure 7(a ) ) .
ap-22 immunofluorescence was observed mainly at the cell periphery , while that for myo6 was also found intracellularly ( figure 6(a ) ) .
regions of overlap were rare and imperfect ( figure 7(a)(ii ) ) , suggesting that the two proteins are not substantially associated within the cells .
experiments using an alexa555-linked secondary antibody for dab2 , however , indicated that there was substantial colocalisation between ap-22 and dab2 ( figure 7(b ) ) .
the compositions ( protein , total cholesterol , and tbars content ) of the nldl and oxldl preparations used are shown in table 3 .
no significant differences were found in the protein or total cholesterol content of nldl as compared to oxldl .
the concentration of tbars , however , was approximately 19-fold higher in oxldl , indicating a significantly increased oxidative state .
thp-1 macrophages were incubated with or without nldl or oxldl ( 50  g protein / ml ) for 8  h or 24  h , and lysates were then used to determine the abundance of transcripts for myo6 , dab2 , ap-22 , and gipc1 by a combination of rt - qpcr and immunoblotting ( the latter for myo6 and dab2 ) .
incubation with nldl caused no significant change in myo6 or dab2 mrna levels at either time point ( figures 8(a ) and 8(b ) ) .
mrna abundance for ap-22 , however , was decreased by about 35% after 24  h ( p < 0.001 ) and that for gipc1 by 31% after 8  h ( p < 0.05 ) or 39% after 24  h ( p < 0.001 ) ( figures 8(c ) and 8(d ) ) .
oxldl , however , caused a marked decrease in mrna levels for myo6 at both time points ( 8  h , 47% , p < 0.01 ; 24  h , 61% , p < 0.05 ) and also significantly reduced mrna levels for ap-22 ( 39% , p < 0.001 ) and gipc1 ( 22% , p < 0.05 ) compared to control values .
myo6 and dab2 protein concentrations showed little change when macrophages were incubated with either nldl or oxldl , with a small decrease in dab2 ( 19% ) in the presence of nldl being the only difference from control values ( figures 8(e)8(h ) ) .
myo6 proteins levels , however , were significantly lower after treatment with oxldl as compared to nldl after 8  h ( + 47% , p < 0.01 , figure 8(f ) ) , while dab2 levels were significantly higher ( + 24% , p < 0.01 , figure 8(h ) ) .
the effects of inhibition of the expression of myo6 , dab2 , ap-22 , and gipc1 on the uptake of nldl and oxldl by thp-1 macrophages were studied using sirna sequences designed to target their cognate genes .
mrna abundance for all test proteins was maximally decreased in thp-1 macrophages 2448  h after transfection with antisense oligonucleotides as compared to scrambled controls ( reduction in abundance was 7585% , myo6 , dab2 , and ap-22 , p < 0.0001 ; 50% gipc , p < 0.001 ) ( figures 9(a)9(d ) ) .
after 72  h ( ap-22 ) or 96  h ( myo6 and dab2 ) , however , the inhibitory effect was reduced for all genes except gipc1 .
in addition , protein concentrations of myo6 were reduced by 7583% between 48 and 96  h and those for dab2 by > 75% between 24 and 96  h ( p < 0.01 ) ( figures 9(e ) and 9(f ) ) .  
uptake of nldl and oxldl by thp-1 macrophages was measured using dii - labelled lipoproteins ( 50  g / ml ) and fluorescence microscopy . in non - sirna - treated cells ,
the area of fluorescence signal increased between 2  h and 24  h , and , as expected , the rate of increase was faster with oxldl as compared to nldl ( figures 10(a ) and 10(b ) ) .
comparison of the areas of fluorescence signal in macrophages transfected with scrambled sirna , or sirna targeting myo6 showed no significant differences ( figures 10(c ) and 10(d ) ) ; similar results were obtained when sirna targeting dab2 , ap-22 or gipc1 were used ( data not shown ) .
myo6 is an intracellular motor protein found to be associated with f - actin in the cytoskeleton [ 7 , 31 ] ; in cells where it functions in endocytosis , it is also found in association with other proteins that have assigned roles in endocytosis , namely , ap-2 , dab2 , and/or gipc1 .
myo6 and the interactive adaptor proteins dab2 , ap-2 , and gipc1 are widely expressed and play diverse , often essential , roles in cellular functioning and signalling .
furthermore , these proteins have been shown to function and interact during cme , and myo6 is thought to provide a driving force for vesicle formation and trafficking . however , although the endocytosis of lipoproteins by macrophages to form foam cells is crucial to atherosclerotic development , the potential roles for myo6 and binding partners in this process have not been explored till now .
indeed , little information is available about the expression of these proteins or their roles in human macrophages , and nothing is known about their subcellular location or mutual interactions in these cells .
we have demonstrated the mrna expression for myo6 , dab2 , ap-2 , and gipc1 in primary human macrophages ( hmdm ) as well as in macrophages derived from the human monocyte cell line , thp-1 ( figure 1 ) .
furthermore , the presence of myo6 , dab2 , and ap-2 protein was demonstrated in these two cell types using both immunoblotting and immunofluorescence ( figures 35 ) .
previous studies have detected dab2 mrna in mouse bone marrow macrophages and myo6 , dab2 , and ap-2 protein in various murine macrophage cell lines [ 1921 , 32 ] .
in addition , myo6 mrna   and low levels of gipc1 mrna   or protein   have been found in human peripheral blood leukocytes . nonetheless , this is the first report of the expression of these proteins in human macrophages , except for one study showing myo6 mrna expression in thp-1 cells .   as positive controls , we demonstrated splice variant expression of myo6 in cos-7 cells , previously shown to express myo6 with and without the li and si insert sequences .
thp-1 macrophages and hmdm were found to express myo6(li ) , but while only one weak band corresponding to myo6(+li ) was observed in cos-7 cells , numerous bands in this region were seen within the two macrophage cell lines ( figures 2(a ) and 2(c ) ) .
dance et al .   found a similar pattern of multiple bands for myo6(+li ) in the epithelial cell lines arpe-19 and llc - pk1 and determined that these resulted from alternative splicing within the li region .
expression of myo6(si ) was demonstrated in thp-1 macrophages and hmdm in our experiments ( figures 2(b ) and 2(d ) ) , but although myo6(+si ) was clearly present in thp-1 cells , its expression was not detected in hmdm .
alternative splice variants of myo6 are differentially expressed and have distinct subcellular locations and functions in various cell types . in polarised epithelial cells , for example , myo6(li )
has been shown to associate with uncoated endocytotic vesicles via gipc1 , while myo6(+li ) is recruited to clathrin coated pits / vesicles via dab2 .
thus , the expression of various myo6 splice forms in human macrophages further indicates that myo6 is likely to have multiple functions , including cme , within these cells . in the current study
, myo6 was found to be distributed throughout the cytoplasmic pool in both thp-1 macrophages and hmdm , with particular localisation to actin - generated protrusions of the plasma membrane , and possibly membrane ruffles ( figure 5 ) , often expressing a punctuate pattern in the cell periphery indicative of association with vesicular structures , as noted previously [ 6 , 14 , 15 ] .
as expected , myo6 appeared to be associated with f - actin , particularly at the apical membrane and at putative podosome attachment sites ( figure 6 ) .
similar associations of myo6 to plasma membrane protrusions and membrane ruffles have been reported in a number of migratory cell types other than macrophages , including epithelial , fibroblast , and ovarian border cells [ 13 , 35 ] , suggesting that it plays an important part in the coordination of the various processes required for cell migration , including cell adhesion and directed cell progression .
chibalina and colleagues   have demonstrated that myo6 functions in endocytosis and vesicle recycling in such cells , and in conjunction with gipc , it has also been shown to participate in the endocytosis of activated 51 integrin , which links it directly to focal adhesion turnover .
have also suggested that the association of myo6 with membrane ruffles in fibroblasts may be indicative of a role in macropinocytosis . during cme , ap-2 is known to act in the formation of clathrin - coated pits / vesicles , while dab2 can either associate with ap-2 as an accessory protein   or act as an adaptor protein in its own right .
dab2 also has numerous functions outside of cme , and so the extent of its association with clathrin - coated vesicles varies between cell types [ 40 , 41 ] . in this study cls fluorescence microscopy was used to show that ap-22 and dab2 ( figure 4 ) are located adjacent to the periphery of both thp-1 macrophages and hmdm in a punctate staining pattern , indicative of vesicle association . to assess the extent of interaction between myo6 , dab2 , and ap-22 in thp-1 macrophages , double immunofluorescence labeling experiments were used .
dab2 and ap-22 were shown to be partially colocalised ( figure 7(b ) ) in a punctate pattern , indicative that dab2 , the direct binding partner of myo6 , is associated with clathrin - coated pits / vesicles in thp-1 macrophages .
this finding is consistent with previous work showing that dab2 is associated with ap-22 in cos-7 cells . however , although association between myo6 and ap-1 has been reported in other cell types , the present experiments suggest that the two proteins do not have substantially overlapping distributions in macrophages ( figure 7(a ) ) .
having shown that myo6 , dab2 , ap-2 , and gipc1 are expressed in human macrophages and that there are some interactions between them , we explored the possibility that they might play a part in macrophage foam cell formation .
thp-1 macrophages were used for these studies since there are serious technical difficulties in obtaining sufficient hmdm for the types of experiment needed .
these cells have some advantages over hmdm in that , because they are genetically homogeneous , there is less variability in their phenotype , and they can be stored indefinitely at 80c .
another factor which was important for our experiments is that transfection efficiency of thp-1 macrophages with sirna is much greater than that with hmdm .
the uptake of nldl and oxldl by macrophages is known to be mediated by different receptors : nldl undergoes endocytosis via the ldl receptor ( ldlr ) which is down - regulated when intracellular cholesterol levels increase , while oxldl is recognised by unregulated scavenger receptors , thus its uptake is the main driver of foam cell formation . since numerous studies have demonstrated that nldl may also directly contribute to foam cell formation [ 4345 ] , both nldl and oxldl were used in our studies .
the expression of the scavenger receptors cd36 , cd68 and scavenger receptor - a ( sr - a ) has been shown to be upregulated in the presence of oxldl and to a lesser extent nldl [ 4649 ] , while ldlr expression is downregulated in response to cell exposure by nldl [ 50 , 51 ] . in the present work , the effects of nldl and oxldl on the expression of mrna and protein for myo6 , dab2 , ap-2 , and gipc1 in thp-1 macrophages were investigated . compared to control levels , nldl significantly decreased mrna transcript abundance for ap-22 and gipc1 ( figures 8(c ) and 8(d ) ) , while incubation with oxldl resulted in reduced levels of myo6 and ap-22 mrna ( figures 8(a ) and 8(c ) ) .
little change was seen at the protein level , however , except for a small decrease in dab2 with nldl ( figures 8(e)8(h ) ) .
both myo6 protein and mrna levels were significantly lower after incubation with oxldl compared to nldl ( figures 8(a ) and 8(f ) ) , suggesting that nldl and oxldl have different effects on the endocytic machinery , possibly reflecting their uptake via different receptor - mediated endocytic routes [ 52 , 53 ] .
we also examined the effects of knockingdown the expression of myo6 and associated proteins on the uptake of nldl and oxldl .
although substantial knockdown of gene expression was achieved ( figure 9 ) , we did not detect any significant effect on the uptake of lipoprotein type as assessed by fluorescence microscopy ( figure 10 ) .
these results , together with our findings of relatively small effects of the lipoproteins on mrna and protein expression ( figure 8) , suggest that myo6 and its binding partners do not play a major role in the uptake of nldl or oxldl by macrophages during induction of foam cell formation .
the results presented here show that myo6 and its associated proteins dab2 , ap-2 , and gipc
are expressed in human macrophages , displaying similar patterns of expression and subcellular location in both the thp-1 cell line and in primary cells ( hmdm ) .
myo6 was found to be particularly prominent in protrusions of the plasma membrane and membrane ruffles , where it was associated with f - actin . ap-2 and dab2 appeared to be associated with cell vesicles , and ap-2 , but not myo6 , was colocalised with dab2 . although nldl and oxldl had some effects on the expression of the genes for the proteins investigated in macrophages , attenuation of their expression through the action of cognate sirna molecules did not affect their uptake by the cells . because the nature of these experiments requires large number of cells , however , hmdm could not be used , thp-1 cells being used instead ; for this reason we remain cautious in extrapolating our findings to primary cells , as would be the case with any study involving immortalized cell lines .
nevertheless , the findings presented here on expression and subcellular localisation suggest that , in macrophages , myo6 functions in cell adhesion and progression as well as in macropinocytosis .
data from the experiments on the uptake of ldl do not support the idea that it plays a major role in foam cell formation .
 myosin vi ( myo6 ) functions in endocytosis in conjunction with binding partners including adaptor protein ( ap)-2 , disabled 2 ( dab2 ) , and gaip interacting protein c terminus 1 ( gipc1 ) . 
 this study aimed to investigate the expression and function of myo6 in macrophages and its possible role in the endocytosis of lipoproteins during the induction of foam cell formation . 
 expression of myo6 , ap-2 ( 2 subunit ) , and dab2 in thp-1 macrophages and primary human monocyte - derived macrophages was demonstrated at the mrna and protein level , but gipc1 was only detected at the mrna level . 
 immunofluorescence showed that myo6 was distributed similarly to f - actin in both macrophage types . 
 ap-22 was found to have a similar subcellular distribution to myo6 and dab2 in thp-1 cells . 
 myo6 was located within membrane ruffles and protrusions of the plasma membrane . 
 these results suggest that in macrophages myo6 is required for several functions including cell adhesion , cell progression , and macropinocytosis . 
 low - density lipoprotein ( ldl ) and oxidised ldl ( oxldl ) decreased myo6 and gipc1 mrna expression in thp-1 cells , but uptake of the fluorescence - labelled lipoproteins was unaffected by knockdown of the expression of myo6 or associated proteins with sirna . 
 our findings , therefore , do not support the idea that myo6 plays a major role in foam cell formation . 