FSGS-associated α-actinin-4 (K256E) impairs cytoskeletal dynamics in podocytes
2006; Elsevier BV; Volume: 70; Issue: 6 Linguagem: Inglês
10.1038/sj.ki.5001665
ISSN1523-1755
AutoresJean Michaud, K.M. Chaisson, Robin J. Parks, C. Kennedy,
Tópico(s)Ovarian cancer diagnosis and treatment
ResumoMutations in the ACTN4 gene, encoding the actin crosslinking protein α-actinin-4, are associated with a familial form of focal segmental glomerulosclerosis (FSGS). Mice with podocyte-specific expression of K256E α-actinin-4 develop foot process effacement and glomerulosclerosis, highlighting the importance of the cytoskeleton in podocyte structure and function. K256E α-actinin-4 exhibits increased affinity for F-actin. However, the downstream effects of this aberrant binding on podocyte dynamics remain unclear. Wild-type and K256E α-actinin-4 were expressed in cultured podocytes via adenoviral infection to determine the effect of the mutation on α-actinin-4 subcellular localization and on cytoskeletal-dependent processes such as adhesion, spreading, migration, and formation of foot process-like peripheral projections. Wild-type α-actinin-4 was detected primarily in the Triton-soluble fraction of podocyte lysates and localized to membrane-associated cortical actin and focal adhesions, with some expression along stress fibers. Conversely, K256E α-actinin-4 was detected predominantly in the Triton-insoluble fraction, was excluded from cortical actin, and localized almost exclusively along stress fibers. Both wild-type and K256E α-actinin-4-expressing podocytes adhered equally to an extracellular matrix (collagen-I). However, podocytes expressing K256E α-actinin-4 showed a reduced ability to spread and migrate on collagen-I. Lastly, K256E α-actinin-4 expression reduced the mean number of actin-rich peripheral projections. Our data suggest that aberrant sequestering of K256E α-actinin-4 impairs podocyte spreading, motility, and reduces the number of peripheral projections. Such intrinsic cytoskeletal derangements may underlie initial podocyte damage and foot process effacement encountered in ACTN4-associated FSGS. Mutations in the ACTN4 gene, encoding the actin crosslinking protein α-actinin-4, are associated with a familial form of focal segmental glomerulosclerosis (FSGS). Mice with podocyte-specific expression of K256E α-actinin-4 develop foot process effacement and glomerulosclerosis, highlighting the importance of the cytoskeleton in podocyte structure and function. K256E α-actinin-4 exhibits increased affinity for F-actin. However, the downstream effects of this aberrant binding on podocyte dynamics remain unclear. Wild-type and K256E α-actinin-4 were expressed in cultured podocytes via adenoviral infection to determine the effect of the mutation on α-actinin-4 subcellular localization and on cytoskeletal-dependent processes such as adhesion, spreading, migration, and formation of foot process-like peripheral projections. Wild-type α-actinin-4 was detected primarily in the Triton-soluble fraction of podocyte lysates and localized to membrane-associated cortical actin and focal adhesions, with some expression along stress fibers. Conversely, K256E α-actinin-4 was detected predominantly in the Triton-insoluble fraction, was excluded from cortical actin, and localized almost exclusively along stress fibers. Both wild-type and K256E α-actinin-4-expressing podocytes adhered equally to an extracellular matrix (collagen-I). However, podocytes expressing K256E α-actinin-4 showed a reduced ability to spread and migrate on collagen-I. Lastly, K256E α-actinin-4 expression reduced the mean number of actin-rich peripheral projections. Our data suggest that aberrant sequestering of K256E α-actinin-4 impairs podocyte spreading, motility, and reduces the number of peripheral projections. Such intrinsic cytoskeletal derangements may underlie initial podocyte damage and foot process effacement encountered in ACTN4-associated FSGS. Focal segmental glomerulosclerosis (FSGS) is a common glomerular lesion and a significant cause of end-stage renal disease.1.Conlon P.J. Butterly D. Albers F. et al.Clinical and pathologic features of familial focal segmental glomerulosclerosis.Am J Kidney Dis. 1995; 26: 34-40Abstract Full Text PDF PubMed Scopus (58) Google Scholar,2.Conlon P.J. Lynn K. Winn M.P. et al.Spectrum of disease in familial focal and segmental glomerulosclerosis.Kidney Int. 1999; 56: 1863-1871Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar Clinically, FSGS patients present with variable levels of proteinuria and a progressive loss of renal function. Pathologically, FSGS is characterized by segmental sclerosis in a proportion of glomeruli, the filtering units of the kidney.3.D'Agati V. The many masks of focal segmental glomerulosclerosis.Kidney Int. 1994; 46: 1223-1241Abstract Full Text PDF PubMed Scopus (290) Google Scholar Accumulating evidence suggests that defects in podocytes initiate processes leading to the degeneration of filtration integrity and the development of sclerotic lesions.4.Schwartz M.M. The role of podocyte injury in the pathogenesis of focal segmental glomerulosclerosis [in process citation].Ren Fail. 2000; 22: 663-684Crossref PubMed Scopus (33) Google Scholar, 5.Barisoni L. Kriz W. Mundel P. et al.The dysregulated podocyte phenotype: a novel concept in the pathogenesis of collapsing idiopathic focal segmental glomerulosclerosis and HIV-associated nephropathy.J Am Soc Nephrol. 1999; 10: 51-61Crossref PubMed Scopus (6) Google Scholar, 6.Shirato I. Sakai T. Kimura K. et al.Cytoskeletal changes in podocytes associated with foot process effacement in Masugi nephritis.Am J Pathol. 1996; 148: 1283-1296PubMed Google Scholar, 7.Gassler N. Elger M. 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The foot processes are endowed with a microfilament-based contractile apparatus composed of actin, myosin-II, α-actinin, talin, paxillin, and vinculin,10.Drenckhahn D. Franke R.P. Ultrastructural organization of contractile and cytoskeletal proteins in glomerular podocytes of chicken, rat, and man.Lab Invest. 1988; 59: 673-682PubMed Google Scholar and are anchored to the glomerular basement membrane via an α3β1-integrin complex.11.Kretzler M. Regulation of adhesive interaction between podocytes and glomerular basement membrane.Microsc Res Techol. 2002; 57: 247-253Crossref PubMed Scopus (72) Google Scholar,12.Adler S. Characterization of glomerular epithelial cell matrix receptors.Am J Pathol. 1992; 141: 571-578PubMed Google Scholar The intricate morphology of the podocyte, coupled to its exposure to distensile forces within the glomerular capillary render these cells susceptible to damage in many nephropathies, including FSGS. The actin bundling α-actinins are members of the spectrin superfamily. Four isoforms have been described (α-actinin-1 to -4).13.Otey C.A. Carpen O. Alpha-actinin revisited: a fresh look at an old player.Cell Motil Cytoskeleton. 2004; 58: 104-111Crossref PubMed Scopus (344) Google Scholar While α-actinins-2 and -3 are expressed at the Z-line of striated muscle, α-actinins-1 and -4 are more ubiquitously expressed. α-Actinin-4 is expressed in podocytes and is thought to play a key role in the maintenance of this cell's architecture.14.Lachapelle M. Bendayan M. Contractile proteins in podocytes: immunocytochemical localization of actin and alpha-actinin in normal and nephrotic rat kidneys.Virchows Arch B. 1991; 60: 105-111Crossref Scopus (40) Google Scholar The putative function of α-actinin-4, which exists as a head-to-tail homodimer, is to crosslink actin filaments through its N-terminal actin-binding domain comprised of two calponin homology domains. Increasing evidence suggests that α-actinin-4 may interact with a number of other proteins such as β1-integrin,13.Otey C.A. Carpen O. Alpha-actinin revisited: a fresh look at an old player.Cell Motil Cytoskeleton. 2004; 58: 104-111Crossref PubMed Scopus (344) Google Scholar, 15.Otey C.A. Pavalko F.M. Burridge K. An interaction between alpha-actinin and the beta 1 integrin subunit in vitro.J Cell Biol. 1990; 111: 721-729Crossref PubMed Scopus (654) Google Scholar, 16.Pavalko F.M. Otey C.A. Simon K.O. et al.Alpha-actinin: a direct link between actin and integrins.Biochem Soc Trans. 1991; 19: 1065-1069Crossref PubMed Scopus (87) Google Scholar synaptodpodin,17.Asanuma K. Kim K. Oh J. et al.Synaptopodin regulates the actin-bundling activity of alpha-actinin in an isoform-specific manner.J Clin Invest. 2005; 115: 1188-1198Crossref PubMed Scopus (264) Google Scholar vinculin,18.Rajfur Z. Roy P. Otey C. et al.Dissecting the link between stress fibres and focal adhesions by CALI with EGFP fusion proteins.Nat Cell Biol. 2002; 4: 286-293Crossref PubMed Scopus (167) Google Scholar and phosphatidylinositol 3-kinase.19.Shibasaki F. Fukami K. Fukui Y. et al.Phosphatidylinositol 3-kinase binds to alpha-actinin through the p85 subunit.Biochem J. 1994; 302: 551-557Crossref PubMed Scopus (89) Google Scholar In addition to an actin-binding domain, a number of other functional domains are found in the α-actinin-4 sequence – including two calcium-binding EF hands, a phosphoinositide-binding domain, as well as a focal adhesion kinase (FAK) tyrosine phosphorylation consensus sequence. Accordingly, phosphorylation by FAK,20.Izaguirre G. Aguirre L. Hu Y.P. et al.The cytoskeletal/non-muscle isoform of alpha-actinin is phosphorylated on its actin-binding domain by the focal adhesion kinase.J Biol Chem. 2001; 276: 28676-28685Crossref PubMed Scopus (120) Google Scholar binding of phosphoinositides,21.Corgan A.M. Singleton C. Santoso C.B. et al.Phosphoinositides differentially regulate alpha-actinin flexibility and function.Biochem J. 2004; 378: 1067-1072Crossref PubMed Scopus (46) Google Scholar,22.Fraley T.S. Tran T.C. Corgan A.M. et al.Phosphoinositide binding inhibits alpha-actinin bundling activity.J Biol Chem. 2003; 278: 24039-24045Crossref PubMed Scopus (74) Google Scholar and sensitivity to intracellular calcium23.Furukawa R. Maselli A. Thomson S.A. et al.Calcium regulation of actin crosslinking is important for function of the actin cytoskeleton in Dictyostelium.J Cell Sci. 2003; 116: 187-196Crossref PubMed Scopus (54) Google Scholar may modulate the actin-binding properties and localization of α-actinin following various environmental stimuli. Mutations in the ACTN4 gene (K228E, T232I, and S235P) are associated with an autosomal-dominant form of FSGS.24.Kaplan J.M. Kim S.H. North K.N. et al.Mutations in ACTN4, encoding alpha-actinin-4, cause familial focal segmental glomerulosclerosis.Nat Genet. 2000; 24: 251-256Crossref PubMed Scopus (1041) Google Scholar,25.Weins A. Kenlan P. Herbert S. et al.Mutational and biological analysis of alpha-actinin-4 in focal segmental glomerulosclerosis.J Am Soc Nephrol. 2005; 16: 3694-3701Crossref PubMed Scopus (142) Google Scholar We developed a mouse model of ACTN4-associated FSGS by expressing the murine correlate of the K228E mutation (K256E) in a podocyte-specific manner using the mNPHS1 promoter.26.Michaud J.L. Lemieux L.I. Dube M. et al.Focal and segmental glomerulosclerosis in mice with podocyte-specific expression of mutant alpha-actinin-4.J Am Soc Nephrol. 2003; 14: 1200-1211Crossref PubMed Scopus (126) Google Scholar These mice exhibit significant proteinuria and develop FSGS-like lesions, confirming that the disease originates in the podocyte. However, the mechanism by which mutations in α-actinin-4 dysregulate podocyte function is not fully understood. Abrogation of α-actinin-4 expression in mice yields severe glomerular disease.27.Kos C.H. Le T.C. Sinha S. et al.Mice deficient in alpha-actinin-4 have severe glomerular disease.J Clin Invest. 2003; 111: 1683-1690Crossref PubMed Scopus (212) Google Scholar Furthermore, recent studies by Yao et al.28.Yao J. Le T.C. Kos C.H. et al.Alpha-actinin-4-mediated FSGS: an inherited kidney disease caused by an aggregated and rapidly degraded cytoskeletal protein.PLoS Biol. 2004; 2: e167Crossref PubMed Scopus (131) Google Scholar suggest that the familial mutations promote α-actinin-4 aggregation and thereby target the protein for degradation via the proteasome pathway, resulting in a partial loss-of-function. In contrast, mutations in α-actinin-4 increase its affinity for filamentous actin (F-actin), suggesting a gain-of-function mechanism.24.Kaplan J.M. Kim S.H. North K.N. et al.Mutations in ACTN4, encoding alpha-actinin-4, cause familial focal segmental glomerulosclerosis.Nat Genet. 2000; 24: 251-256Crossref PubMed Scopus (1041) Google Scholar,26.Michaud J.L. Lemieux L.I. Dube M. et al.Focal and segmental glomerulosclerosis in mice with podocyte-specific expression of mutant alpha-actinin-4.J Am Soc Nephrol. 2003; 14: 1200-1211Crossref PubMed Scopus (126) Google Scholar In support of the latter mechanism, the severity of the FSGS-like phenotype correlates directly with K256E α-actinin-4 levels in transgenic mice.26.Michaud J.L. Lemieux L.I. Dube M. et al.Focal and segmental glomerulosclerosis in mice with podocyte-specific expression of mutant alpha-actinin-4.J Am Soc Nephrol. 2003; 14: 1200-1211Crossref PubMed Scopus (126) Google Scholar Thus, it remains unclear whether and how these two viewpoints can be reconciled. To address this issue, we assessed the functional consequences of an FSGS-associated mutation (K256E) in α-actinin-4 at the cellular level. We now report the intracellular mislocalization of K256E α-actinin-4 in mouse podocytes, which undermines the processes of cell spreading and migration, and impairs the formation of actin-rich peripheral projections. Our data suggest that such defects in key cytoskeletal-associated processes may compromise the podocyte's ability to cope with the demands of the glomerular environment, maintain foot processes structure, and thereby initiate the progression towards sclerosis. Mutations in α-actinin-4 increase its affinity for F-actin in vitro.24.Kaplan J.M. Kim S.H. North K.N. et al.Mutations in ACTN4, encoding alpha-actinin-4, cause familial focal segmental glomerulosclerosis.Nat Genet. 2000; 24: 251-256Crossref PubMed Scopus (1041) Google Scholar,26.Michaud J.L. Lemieux L.I. Dube M. et al.Focal and segmental glomerulosclerosis in mice with podocyte-specific expression of mutant alpha-actinin-4.J Am Soc Nephrol. 2003; 14: 1200-1211Crossref PubMed Scopus (126) Google Scholar However, the subcellular localization of mutant α-actinin-4 is not clearly defined. We therefore generated adenoviral constructs with hemagglutinin (HA)-tagged wild-type or K256E α-actinin-4 and transduced a conditionally immortalized mouse podocyte cell line. Podocytes were infected with a range of virus to determine a concentration yielding efficient expression (Figure 1a). For all subsequent experiments, infections were performed with a multiplicity of infection (MOI) of 25 and incubated for 72 h. There was no apparent degradation of heterologously expressed K256E α-actinin-4 during this timeframe as its expression paralleled that of the wild-type protein (Figure 1a). As shown in Figure 1b, wild-type α-actinin-4 localized predominantly with cortical actin. The wild-type protein was also distributed along stress fibers and at focal adhesions, as identified by vinculin co-immunofluorescence (Figure 1c). Conversely, K256E α-actinin-4 was absent from the cell periphery, but was preferentially associated with stress fibers (Figure 1b) and focal adhesions (Figure 1c). We next performed cellular fractionation experiments to determine the association of wild-type and K256E α-actinin-4 with various intracellular actin pools. Podocytes expressing wild-type or K256E α-actinin-4 were lysed in Triton X-100-containing buffer and subject to differential centrifugation. As shown in Figure 2a and b, only 16.0±2.5% of the wild-type α-actinin-4 associated with large cytoskeletal structures (Triton X-100 insoluble (TI) fraction), whereas 71.4±5.5% of the protein remained soluble (Triton X-100 soluble (TS):S fraction). Conversely, 81.0±9.6% of the K256E α-actinin-4 was associated with large cytoskeletal structures (TI fraction), and only 4.9±3.4% of the mutant protein remained soluble (TS:S fraction). Expression of both wild-type and K256E α-actinin-4 was similar, as evidenced by the input. Furthermore, neither the expression of wild-type nor K256E α-actinin-4 altered total actin levels. These data reveal a differential association of wild-type versus K256E α-actinin-4, with K256E α-actinin-4 sequestered to large cytoskeletal structures such as actin bundles, whereas wild-type α-actinin-4 remains predominantly soluble. The inappropriate association of K256E α-actinin-4 with the actin cytoskeleton suggested that it may negatively affect cytoskeletal dynamics. We therefore determined its effect on cytoskeletal-dependent processes, such as cell adhesion, spreading, and migration. Since α-actinin-4 is associated with focal adhesions, we hypothesized that the mutant protein may negatively affect the ability of cells to adhere to an extracellular matrix. Adhesion assays were performed using podocytes expressing green fluorescent protein (GFP) alone (control), wild-type α-actinin-4, or K256E α-actinin-4 measuring their ability to bind to collagen-I-coated wells (Figure 3). The number of adherent cells was quantified at measuring various time points (3–24 h). Irrespective of the time allowed for adhesion, there was no difference in adhesion between wild-type and K256E α-actinin-4-expressing podocytes, suggesting that cell–matrix interactions are not adversely affected by K256E α-actinin-4. We next performed a replating assay to assess the ability of cells expressing either wild-type or K256E α-actinin-4 to efficiently spread on an extracellular matrix (collagen-I). Podocytes expressing GFP alone (control), wild-type, or K256E α-actinin-4 were harvested and replated onto collagen-I-coated glass coverslips. Adherent cells were fixed after 3 or 6 h and visualized by immunofluorescence. Within 3 h of replating, a significant number of podocytes had adhered to the substratum and had begun to spread. For each condition, we observed no differences in the total number of podocytes adhering to the extracellular matrix. However, at both time points examined, the number of spreading podocytes was significantly lower in K256E α-actinin-4-expressing cells (13.0±0.3% at 3 h; 17.3±1.0% at 6 h) compared to control (29.2±5.8% at 3 h; 37.7±5.2% at 6 h) and wild type (34.7±4.4% at 3 h; 38.4±5.8% at 6 h) (Figure 4a). Within 6 h of replating, wild-type α-actinin-4 was localized with cortical actin at the cell periphery (Figure 4b). Conversely, K256E α-actinin-4 remained associated with F-actin and condensed in the cell body (Figure 4b), consistent with the observed impairment in cell spreading. Cell migration relies upon a dynamic cytoskeleton. The increased affinity of K256E α-actinin-4 for F-actin could undermine this process. We therefore performed haptotactic transwell migration assays to determine the effect of K256E α-actinin-4 on cell migration. Podocytes expressing GFP alone (control), wild-type, or K256E α-actinin-4 were plated onto the upper surface of transwell inserts. After 24 h, the cells remaining in the upper chamber were removed and the cells that had migrated to the underside of the insert were quantified. Podocytes expressing wild-type α-actinin-4 migrated at a similar rate (90.8±9.2%) to control cells (Figure 5). Conversely, the migration of podocytes expressing K256E α-actinin-4 was significantly reduced (52.1±10.7%; P<0.01 vs control and wild type). These data suggest that K256E α-actinin-4 causes defects in cytoskeletal dynamics and impairs cellular processes such as spreading and migration. Podocyte-specific expression of K256E α-actinin-4 leads to podocyte damage and foot process effacement in vivo.26.Michaud J.L. Lemieux L.I. Dube M. et al.Focal and segmental glomerulosclerosis in mice with podocyte-specific expression of mutant alpha-actinin-4.J Am Soc Nephrol. 2003; 14: 1200-1211Crossref PubMed Scopus (126) Google Scholar The conditionally immortalized podocyte cell line used in the present studies have been shown to form foot process-like peripheral projections when cultured under non-permissive conditions.29.Mundel P. Reiser J. Zuniga Mejia Borja A. et al.Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines.Exp Cell Res. 1997; 236: 248-258Crossref PubMed Scopus (767) Google Scholar We therefore assessed the effect of K256E α-actinin-4 on peripheral projections in differentiated podocytes (Figure 6). Podocytes expressing wild-type α-actinin-4 displayed a slight increase in the mean number of projections (3.8±0.8 projections/cell vs 2.9±0.6 for control), whereas podocytes expressing K256E α-actinin-4 exhibited a decrease in the mean number of projections (1.9±0.4) (Figure 6b). Wild-type but not K256E α-actinin-4 was readily detected in the peripheral projections, along with actin (Figure 6a). Furthermore, projections emerging from podocytes expressing wild-type α-actinin-4 appeared longer and thinner than those of control and K256E α-actinin-4-expressing podocytes. These data provide evidence that α-actinin-4 plays a key role in the formation and/or maintenance of peripheral projections in cultured podocytes. We previously developed a mouse model of an inherited form of FSGS by expressing a mutant variant of α-actinin-4 (K256E) under the control of a podocyte-specific promoter (mNPHS1).26.Michaud J.L. Lemieux L.I. Dube M. et al.Focal and segmental glomerulosclerosis in mice with podocyte-specific expression of mutant alpha-actinin-4.J Am Soc Nephrol. 2003; 14: 1200-1211Crossref PubMed Scopus (126) Google Scholar In this model, approximately 50% of mice develop FSGS-like lesions and display podocyte foot process effacement. However, the direct consequences of mutant α-actinin-4 on podocyte structure and function remained unclear. We therefore assessed the effects of K256E α-actinin-4 on the cytoskeletal dynamics of cultured podocytes. The conditionally immortalized podocyte cell line used in this study has previously been described in detail.29.Mundel P. Reiser J. Zuniga Mejia Borja A. et al.Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines.Exp Cell Res. 1997; 236: 248-258Crossref PubMed Scopus (767) Google Scholar These cells are relatively resistant to conventional transfection approaches for achieving heterologous expression of proteins. We therefore developed adenoviruses for both wild-type and K256E α-actinin-4, which yielded significant expression in a high proportion of cells (90%), rendering the cell population much more homogeneous than previously attained. The expression level of both wild-type and mutant K256E α-actinin-4 were similar (Figure 1a), suggesting that the mutant protein is not subject to degradation in these cells. The most impressive feature upon expression of K256E α-actinin-4 is its distinct intracellular localization. Although the putative function of α-actinin-4 is that of an actin crosslinking protein, we found that the majority of wild-type α-actinin-4 colocalized with membrane-associated cortical actin (Figure 1b) or was detected in the TS pool (Figure 2). In contrast, K256E α-actinin-4 was consistently found along stress fibers and was retained in the TI fraction (Figure 2). This sequestration is likely a direct effect of the increased affinity of K256E α-actinin-4 for F-actin, and is consistent with a gain-of-function effect of such mutations. In support of this, an alternative splice variant of α-actinin-4 has been reported in small-cell lung cancer.30.Honda K. Yamada T. Seike M. et al.Alternative splice variant of actinin-4 in small cell lung cancer.Oncogene. 2004; 23: 5257-5262Crossref PubMed Scopus (47) Google Scholar The splice variant contains three missense mutations in exon 8, the region containing FSGS-associated mutations. This mutant isoform also displays increased affinity for actin, and was found to colocalize mainly with actin stress fibers, unlike the wild-type protein, which was concentrated along the periphery of the cell. Several reports have identified an association of actinin isoforms with components of focal adhesion complexes, such as the β1-integrin subunit and vinculin.13.Otey C.A. Carpen O. Alpha-actinin revisited: a fresh look at an old player.Cell Motil Cytoskeleton. 2004; 58: 104-111Crossref PubMed Scopus (344) Google Scholar Phosphoinositides may also bind to α-actinin and regulate its interaction with actin filaments or integrin receptors.21.Corgan A.M. Singleton C. Santoso C.B. et al.Phosphoinositides differentially regulate alpha-actinin flexibility and function.Biochem J. 2004; 378: 1067-1072Crossref PubMed Scopus (46) Google Scholar,22.Fraley T.S. Tran T.C. Corgan A.M. et al.Phosphoinositide binding inhibits alpha-actinin bundling activity.J Biol Chem. 2003; 278: 24039-24045Crossref PubMed Scopus (74) Google Scholar Furthermore, α-actinin is phosphorylated by FAK in platelets,20.Izaguirre G. Aguirre L. Hu Y.P. et al.The cytoskeletal/non-muscle isoform of alpha-actinin is phosphorylated on its actin-binding domain by the focal adhesion kinase.J Biol Chem. 2001; 276: 28676-28685Crossref PubMed Scopus (120) Google Scholar reducing its cosedimentation with actin filaments. These findings clearly indicate a role for α-actinin in mediating signals that could modulate the assembly of focal adhesions or the cytoskeleton. It is therefore interesting to speculate that mutations in α-actinin-4, which increase its affinity for actin, may render the protein insensitive to these factors and thereby perturb cytoskeletal dynamics. The mislocalization of mutant α-actinin-4 led us to investigate its effects on cytoskeletal-dependent processes such as cell adhesion, spreading, and migration. Although we did not observe a significant impact of K256E α-actinin-4 on podocyte adhesion (i.e., adhesion and replating assays), we found that it significantly affected spreading and migration. Indeed, podocytes expressing K256E α-actinin-4 remained rounded and showed severe defects in their ability to spread on collagen-I. This phenotype is reminiscent of that reported in FAK-deficient embryonic mesodermal cells,31.Ilic D. Furuta Y. Kanazawa S. et al.Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice.Nature. 1995; 377: 539-544Crossref PubMed Scopus (1586) Google Scholar which can adhere but fail to spread effectively on ECM, suggesting that FAK signaling may be impaired in cells expressing mutant α-actinin-4. Furthermore, as indicated by the present study, podocytes expressing K256E α-actinin-4 exhibit severe motility defects. These findings are in accordance with a role of actinin-4 in cell motility and cancer invasion,32.Honda K. Yamada T. Endo R. et al.Actinin-4, a novel actin-bundling protein associated with cell motility and cancer invasion.J Cell Biol. 1998; 140: 1383-1393Crossref PubMed Scopus (390) Google Scholar, 33.Menez J. Le Maux Chansac B. Dorothee G. et al.Mutant alpha-actinin-4 promotes tumorigenicity and regulates cell motility of a human lung carcinoma.Oncogene. 2004; 23: 2630-2639Crossref PubMed Scopus (38) Google Scholar, 34.Honda K. Yamada T. Hayashida Y. et al.Actinin-4 increases cell motility and promotes lymph node metastasis of colorectal cancer.Gastroenterology. 2005; 128: 51-62Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar where cytoplasmic localization of α-actinin-4 is associated with an infiltrative phenotype in breast cancer cells, whereas cells with nuclear α-actinin-4 are non-invasive. Our results show that α-actinin-4 is significantly involved in cell spreading and migration. The motility deficit seen in cells expressing K256E α-actinin-4 is likely owing to the sequestering of the protein to centrally located actin stress fibers, and away from motility-based structures at the cell's periphery. Expression of wild-type α-actinin-4 increased the mean number of actin filament-containing peripheral projections emanating from the cell body, reminiscent of podocyte foot processes in vivo. In contrast, K256E α-actinin-4 reduced the mean number of such projections (Figure 6). Although the arborized phenotype observed in cultured podocytes is not nearly as extensive as that seen in podocytes in vivo,35.Kobayashi N. Reiser J. Schwarz K. et al.Process formation of podocytes: morphogenetic activity of microtubules and regulation by protein serine/threonine phosphatase PP2A.Histochem Cell Biol. 2001; 115: 255-266PubMed Google Scholar our data suggest that owing to mislocalization, K256E α-actinin-4 does not provide a suitable framework for the maintenance/formation of such projections. We previously showed that podocyte-specific expression of K256E α-actinin-4 leads to foot process effacement and glomerulosclerosis. Such damage may be explained by mislocalization of mutant α-actinin-4, rendering it unresponsive to appropriate signals (e.g., actin remodeling) or unable to provide structural support at the cell membrane, and thereby contribute to foot process effacement. A recent study by Yao et al.28.Yao J. Le T.C. Kos C.H. et al.Alpha-actinin-4-mediated FSGS: an inherited kidney disease caused by an aggregated and rapidly degraded cytoskeletal protein.PLoS Biol. 2004; 2: e167Crossref PubMed Scopus (131) Google Scholar attributes podocyte defects encountered in ACTN4-associated FSGS to a loss-of-function as mutations render α-actinin-4 more susceptible to forming unstable aggregates, which are rapidly degraded via the proteasome pathway. Conversely, in transgenic mice expressing an FSGS-associated mutant α-actinin-4, podocyte damage and sclerosis correlate directly with mutant transgene expression, consistent with a gain-of-function mechanism.26.Michaud J.L. Lemieux L.I. Dube M. et al.Focal and segmental glomerulosclerosis in mice with podocyte-specific expression of mutant alpha-actinin-4.J Am Soc Nephrol. 2003; 14: 1200-1211Crossref PubMed Scopus (126) Google Scholar We did not observe any degradation of exogenous K256E α-actinin-4 in cultured podocytes (Figure 1a) within the experimental timeframe. In light of these findings, we favor a synthesis of the two models to explain the dysregulated phenotype of podocytes expressing FSGS-associated mutant α-actinin-4. Gain-of-affinity mutations in α-actinin-4, coupled with some degradation of the mutant protein, could each contribute to a loss-of-function by effectively eliminating this protein from select intracellular locales (e.g., motility-based actin structures and process-like peripheral projections), thereby disturbing cytoskeletal-dependent processes such as cell spreading, migration, and importantly, process maintenance/formation. In support of this hypothesis, dysregulation of the actin cytoskeleton and α-actinin-4 expression/localization has been reported in various glomerular disease models characterized by podocyte foot process effacement,6.Shirato I. Sakai T. Kimura K. et al.Cytoskeletal changes in podocytes associated with foot process effacement in Masugi nephritis.Am J Pathol. 1996; 148: 1283-1296PubMed Google Scholar, 36.Smoyer W.E. Mundel P. Gupta A. et al.Podocyte alpha-actinin induction precedes foot process effacement in experimental nephrotic syndrome.Am J Physiol. 1997; 273: F150-F157PubMed Google Scholar, 37.Smoyer W.E. Mundel P. Regulation of podocyte structure during the development of nephrotic syndrome.J Mol Med. 1998; 76: 172-183Crossref PubMed Scopus (141) Google Scholar, 38.Barisoni L. Kopp J.B. Modulation of podocyte phenotype in collapsing glomerulopathies.Microsc Res Techol. 2002; 57: 254-262Crossref PubMed Scopus (38) Google Scholar and is therefore likely to be a key event in the progression of podocyte injury. In summary, we have further defined the functional consequences of an FSGS-associated form of the actin-crosslinking protein – K256E α-actinin-4. Whereas wild-type α-actinin-4 is predominantly localized to membrane-associated cortical actin in conditionally immortalized podocytes, K256E α-actinin-4 remains tightly associated with stress fibers. Mislocalization of K256E α-actinin-4 causes severe defects in motility, including cell spreading and migration. Furthermore, K256E α-actinin-4 caused a decrease in the number of foot process-like peripheral projections. Our data therefore suggest that dysregulation of the podocyte cytoskeleton may play a key role in the progression of sclerotic lesions resulting from various podocyte injuries. Adenoviral constructs containing wild-type or K256E murine HA-ACTN4 sequences were generated by subcloning from the previously described pcDNA3-HA-ACTN4 vectors.26.Michaud J.L. Lemieux L.I. Dube M. et al.Focal and segmental glomerulosclerosis in mice with podocyte-specific expression of mutant alpha-actinin-4.J Am Soc Nephrol. 2003; 14: 1200-1211Crossref PubMed Scopus (126) Google Scholar In these viruses, the HA-ACTN4 expression cassette replaced the E1-region and transcription is directed rightward, relative to the conventional human adenovirus serotype 5 map. The E1-deleted, first-generation Adenovirus vectors used in these studies were constructed using a combination of conventional cloning techniques and RecA-mediated recombination,39.Chartier C. Degryse E. Gantzer M. et al.Efficient generation of recombinant adenovirus vectors by homologous recombination in Escherichia coli.J Virol. 1996; 70: 4805-4810Crossref PubMed Google Scholar,40.He T.C. Zhou S. da Costa L.T. et al.A simplified system for generating recombinant adenoviruses.Proc Natl Acad Sci USA. 1998; 95: 2509-2514Crossref PubMed Scopus (3250) Google Scholar and were grown and titered on 293 cells, as described previously.41.Ng P. Graham F.L. Construction of first-generation adenoviral vectors.Methods Mol Med. 2002; 69: 389-414PubMed Google Scholar Conditionally immortalized mouse podocytes were cultured on collagen-I-coated dishes in RPMI-1640 supplemented with 10% fetal bovine serum, 100 U/ml of penicillin, 100 μg/ml of streptomycin, 100 μg/ml of Normocin (InvivoGen, San Diego, CA, USA), and 10 U/ml γ-interferon (Sigma, Oakville, Ontario, Canada) at 33°C (permissive conditions). For differentiation, cells were cultured at 38°C without γ-interferon (non-permissive conditions) for at least 10 days. Cells used for experiments were between passages 5 and 15 only. Differentiated podocytes were infected with a range of viral loads (multiplicity of infection between 0 and 50 plaque-forming units/cell) to determine optimal infection conditions. Lysates (10 μg total protein) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Immunoblots were incubated with a monoclonal anti-HA antibody (HA-7, Sigma, 1:1000), followed by a horseradish peroxidase-conjugated secondary antibody and developed using Supersignal West Pico Chemiluminescence (Pierce, Rockford, IL, USA). Immunoblots were stripped and reprobed with an anti-actin antibody (Sigma, 1:1000). For all subsequent experiments, differentiated podocytes were infected at a multiplicity of infection of 25 plaque-forming units/cell and incubated for 72 h to achieve sufficient protein expression. At 3 days post-infection, podocytes grown on collagen-coated coverslips were processed for immunofluorescence. Cells were fixed with 4% paraformaldehyde, washed with phosphate-buffered saline (PBS), permeabilized with 0.2% Triton X-100, blocked with 2% bovine serum albumin, and incubated with an anti-HA tag antibody (Inter Medico, Markham, Ontario, Canada, 1:200 or HA-7, Sigma, 1:1000). Cells were then double-labeled with appropriate Alexa Fluor-conjugated secondary antibodies and Alexa Fluor-conjugated phalloidin (both form Molecular Probes, Eugene, OR, USA, 1:1000). To visualize focal adhesions, cells were labeled with an anti-vinculin antibody (Vin11.5, Sigma, 1:100) followed by appropriate Alexa Fluor-conjugated secondary antibodies and phalloidin. Images were captured using a Zeiss AxioCam and a Zeiss Axioskop 2 fluorescence microscope (Zeiss Axioskop 2 MOT, Zeiss, Germany). Infected differentiated mouse podocytes were scraped into ice-cold lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM ethyleneglycol tetraacetate, 1 mM ethylenediaminetetraacetic acid, 1% Triton X-100, 0.5% Nonidet P-40) supplemented with protease inhibitor cocktail (Sigma, 1:100) and phenylmethylsulfonyl fluoride, and incubated at room temperature for 15 min. Cell lysates were then centrifuged at 15 000 g to isolate the TI fraction. The TS fraction was removed and further centrifuged at 100 000 g to separate G-actin (supernatant, S) and F-actin (pellet, P). All pellets were resuspended in the original sample volume. A small aliquot of the total lysate was reserved for protein determination using a Bicinchoninic Acid Protein Assay Kit (Pierce). Samples of equal volume (10 μg total protein in lysate) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were probed with a polyclonal anti-HA tag antibody (Clontech, Palo Alto, CA, USA, 1:1000) followed by a horseradish peroxidase-conjugated secondary antibody and developed using Supersignal West Pico Chemiluminescent Substrate (Pierce). The immunoblots were subsequently stripped and reprobed with an anti-actin antibody (Sigma, 1:1000). The membranes were exposed to film, images were scanned with a Hewlett Packard ScanJet 6100C, and densitometry of the bands measured using Kodak 1D 3.5 software. Differentiated mouse podocytes, having been infected with adenovirus 72 h earlier, were harvested by trypsin/ethylenediaminetetraacetic acid treatment, centrifuged, and resuspended in RPMI containing 10% fetal bovine serum and counted with a hemacytometer. Cells were seeded onto collagen-I-coated U-bottom 96-well plates (5 × 104 cells/well) and incubated at 38°C. At various times after seeding (3, 6, 12, and 24 h), the wells were washed with PBS and the cells fixed with 4% paraformaldehyde for 30 min at room temperature. Adherent cells were then stained with 1% crystal violet for 30 min, washed five times with PBS, and solubilized in 20% acetic acid. The absorbance (595 nm) of each well was then measured using a FLUOstar Galaxy plate reader. Data are from three separate experiments and are expressed as a function of control cells. Differentiated mouse podocytes, having been infected with adenovirus 72 h earlier, were harvested by trypsin/ethylenediaminetetraacetic acid treatment. Following trypsin inactivation with soybean trypsin inhibitor (Sigma, 0.5 mg/ml), the cells were collected by centrifugation, washed once, and held in suspension for 15 min at 38°C in RPMI containing 10% fetal bovine serum. Suspended cells were counted with a hemacytometer, seeded onto collagen-I-coated glass coverslips (5 × 104 cells/well), and incubated at 38°C. At 3 and 6 h following replating, the wells were washed with PBS, and adherent cells fixed with 4% paraformaldehyde and processed for immunofluorescence as above. The number of spreading cells was counted (three separate experiments) and is expressed as a percentage of infected cells. Differentiated mouse podocytes, having been infected with adenovirus 72 h earlier, were harvested by trypsin/ethylenediaminetetraacetic acid treatment. Following trypsin inactivation with soybean trypsin inhibitor (0.5 mg/ml), cells were collected by centrifugation and resuspended in RPMI +10% fetal bovine serum. Suspended cells were counted by hemacytometer and seeded onto the upper chamber of transwell inserts (5 × 104 cells/insert) pre-coated on the lower surface with collagen-I. After a 24 h incubation at 38°C, cells on the upper surface were removed with a cotton-tip applicator and the migrating cells on the lower surface of the insert were washed with PBS, fixed with paraformaldehyde, and stained with 4′,6-diamidino-2-phenylindole. Membranes were removed from the inserts, placed on slides, and migrating cells were quantified by counting 4′,6-diamidino-2-phenylindole-stained nuclei. Data are from five experiments expressed as the percentage of control. Differentiated podocytes grown on collagen-I-coated coverslips were infected with adenoviruses for wild-type or K2565E α-actinin-4 and processed for immunofluorescence as described above (Fluorescence microsocopy). Peripheral projections emanating from the cell body of HA-positive cells were quantified in a minimum of 50 cells from four experiments. Data are the mean number of projections per cell. Uninfected cells served as controls. Values reported are the means±s.e.m. from at least three experiments. Statistical comparisons were made using unpaired t-test or one-way analysis of variance followed by the Newman–Keuls multiple comparison test. We are grateful to Peter Mundel (Albert Einstein, New York) for the conditionally immortalized podocyte cell line. Robin J Parks is supported by the Canadian Institutes of Health Research (CIHR), CIHR/Muscular Dystrophy Association of Canada/Amyotrophic Lateral Sclerosis Society of Canada Partnership Grant, Premier's Research Excellence Award, and the Jesse Davidson Foundation for Gene and Cell Therapy. CRJ Kennedy and RJ Parks are CIHR New Investigator Awardees. Jean-Louis R Michaud is a recipient of a CIHR Doctoral Award. This work is supported by an operating grant from the Kidney Foundation of Canada.
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