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Actin up: regulation of podocytestructure and function by componentsof the actin cytoskeletonChristian Faul1, Katsuhiko Asanuma2, Etsuko Yanagida-Asanuma2, Kwanghee Kim1

and Peter Mundel1

1 Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA2 Division of Nephrology, Juntendo University School of Medicine, Tokyo, 113-8421, Japan

Review TRENDS in Cell Biology Vol.xxx No.x

Podocytes of the renal glomerulus are unique cells with acomplex cellular organization consisting of a cell body,major processes and foot processes. Podocyte foot pro-cesses form a characteristic interdigitating pattern withfoot processes of neighboring podocytes, leaving inbetween the filtration slits that are bridged by the glo-merular slit diaphragm. The highly dynamic foot pro-cesses contain an actin-based contractile apparatuscomparable to that of smooth muscle cells or pericytes.Mutations affecting several podocyte proteins lead torearrangement of the actin cytoskeleton, disruption ofthe filtration barrier and subsequent renal disease. Thefact that the dynamic regulation of the podocyte cytos-keleton is vital to kidney function has led to podocytesemerging as an excellent model system for studyingactin cytoskeleton dynamics in a physiological context.

IntroductionThe kidney glomerulus is a highly specialized structurethat ensures the selective ultrafiltration of plasma so thatessential proteins are retained in the blood [1]. The glo-merular basement membrane (GBM) provides the primarystructural support for the glomerular tuft. The basic unit ofthe glomerular tuft is a single capillary. Endothelial andsmooth muscle-like mesangial cells providing capillarysupport are located inside the GBM, whereas podocytesare attached to the outer aspect of the GBM (Figure 1a).Human genetic studies revealed that mutations affectingseveral podocyte proteins, including a-actinin-4 [2],nephrin [3], the phospholipase C epsilon gene [4], podocin[5] and transient receptor potential cation channel 6(TRPC6) [6,7], lead to renal disease owing to the disruptionof the filtration barrier and rearrangement of the actincytoskeleton. Cell biological and mouse genetic studiesrevealed that additional proteins regulating the plasticityof the podocyte actin cytoskeleton, such as Rho guaninedissociation inhibitor (GDI)-a [8], podocalyxin [9], FAT1[10], Nck1 and Nck2 [11,12] and synaptopodin (Synpo)[13,14], are also of crucial importance for the sustainedfunction of the glomerular filtration barrier. Although it isimportant to define basic cellular mechanisms in the

Corresponding author: Mundel, P. (peter.mundel@mssm.edu).Available online xxxxxx.

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widest possible terms to understand general cell behavior,some of the human disease-related mechanisms have to bestudied in the appropriate physiological context. Thedynamic regulation of the podocyte cytoskeleton is para-mount for proper kidney filtration. In the context of pro-teinuric kidney diseases, it is necessary to study theregulation of the actin cytoskeleton directly in podocytesbecause the dysregulation of the highly specialized podo-cyte actin cytoskeleton is closely associated with a diseasephenotype. Most of what we know about the regulation ofthe actin cytoskeleton has come from commonly usedgeneric cell culture models, such as HEK293, NIH3T3,COS-7 and HeLa cells, but studies in highly specializedcells such as podocytes has added invaluable cell type-specific and disease-related information. Here, we discussrecent findings shedding new light on the dynamic regu-lation of the podocyte actin cytoskeleton.

Podocytes are pericyte-like cells with an actin-basedcontractile apparatusDifferentiated podocytes are mesenchymal-like cells thatarise from epithelial precursors during renal development(Box 1). They consist of three morphologically and func-tionally different segments: a cell body, major processesand foot processes (FPs) [15]. Major processes arise fromthe cell body and split into FPs (Figure 1a,b). The FPscontain an actin-based cytoskeleton that is linked to theGBM in focal contacts (Figures 1c,e,f and 2). Podocyte FPsform a highly branched interdigitating networkwith FPs ofneighboring podocytes connected by the slit diaphragm(SD) (Figure 1c,d), a multiprotein complex similar to adhe-rens junctions which covers the filtration slits (regionsbetween opposing podocyte FPs) [15], thereby establishingthe final barrier to urinary protein loss [1]. FPs are furthercharacterized by a podosome-like, cortical network of shortbranched actin filaments and the presence of highlyordered parallel (Figure 1d,e), contractile (Figures 2,3a)actin filament bundles [16], which are thought to modulatethe permeability of the filtration barrier through changesin FP morphology. The function of podocytes is largelybased on their complex cell architecture, in particular onthe maintenance of the normal FP structure. FPs arefunctionally defined by three membrane domains(Figure 3b): the apical membrane domain (AMD), the

d. doi:10.1016/j.tcb.2007.06.006

d function by components of the actin cytoskeleton, Trends Cell Biol. (2007),

Figure 1. Podocytes are pericyte-like cells with a complex cytoarchitecture. (a) The GBM provides the primary structural support for the glomerular tuft. Glomerular

endothelial cells (E) embracing the capillary lumen (CL) and mesangial cells (M) are located on the blood side of the GBM, whereas podocytes cover the outer aspect of the

GBM facing Bowman’s space (BS). Podocytes consist of a cell body (green), major processes (MPs) and FPs (red). Only FPs are in direct contact with the GBM. They also

interdigitate with FPs of neighboring cells and form the filtration slits. (b) Scanning electron microscopy illustrates the complexity of podocyte morphology. Looking from

Bowman’s space, a large cell body (CB) is seen that is linked to the capillaries by MPs. FPs arise from MPs and form the signature interdigitating pattern with FPs of a

neighboring podocyte, leaving in between the filtration slits. (c) Transmission electron micrograph of an MP and a segment of the glomerular filtration barrier consisting of

fenestrated endothelium (E), GBM and podocyte FPs with the interposed SD, which cover the filtration slits. The cords connecting the lamina densa of the GBM with

podocyte and endothelial cell membranes represent proteoglycans. (d) The origin of several FPs from a major process is shown. The cytoskeleton of MPs predominantly

consists of microtubules and intermediate filaments. FPs contain bundles of parallel actin filaments (arrows) that connect neighboring FPs. (e) At higher magnification

(�65 000), the well-organized parallel actin bundles (arrows) are clearly visible. In addition, multiple endocytotic vesicles are encountered, both in FPs and MPs. (f) Top view

of normal (i) and effaced (ii) podocyte FPs. In healthy podocytes, FPs regularly interdigitate. The highly organized actin bundles of interdigitating FPs from two adjacent

podocytes are shown in red and yellow, respectively. Microtubules of MPs are shown in blue. By contrast, a meandering cell border is seen between effaced FPs. A

continuous sheet of cytoplasm develops that is filled with reorganized, short, branched actin filaments (green).

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SD and the basal membrane domain (BMD, also known asthe sole plate, which is associated with the GBM). Allthree domains are physically and functionally linked tothe FP actin cytoskeleton, making actin the common den-ominator in podocyte function and dysfunction [17,18].Interference with any of the three FP domains changesthe actin cytoskeleton from parallel contractile bundles[16] into a dense network with FP effacement (reflected bythe simplification of the FP structure and the loss of thenormal interdigitating pattern) (Figure 1f) and proteinuria[17]. FP effacement requires the active reorganization ofactin filaments [9,19]. Therefore, proteins regulatingthe plasticity of the podocyte actin cytoskeleton are ofcrucial importance for themaintenance of glomerular filterfunction.

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The SD is a highly complex multiprotein signaltransduction unitThe SD represents a modified adherens junction [20](Table 1), a complex signal transduction unit composedof several proteins (Figure 3c), which spans the 30–50 nmfiltration slits. The extracellular portion of the SD ismade up of rod-like units, presumably containing theextracellular domains of various transmembrane proteins(Figure 3c). These units are connected in the center to alinear bar, forming a zipper-like pattern, with pores thesame size as, or smaller than, albumin [15]. Interestingly,the cytoplasmic insertion site of the SD is a regionof Triton-X-100-resistant [15] electron dense material(Figure 3a; arrows) which bears a strong morphologicaland biochemical similarity to another highly insoluble

d function by components of the actin cytoskeleton, Trends Cell Biol. (2007),

Box 1. Podocyte differentiation involves a physiological

epithelial–mesenchymal transition

Glomerular development is generally divided into four stages: renal

vesicle stage, S-shaped body stage, capillary loop stage and

maturing glomeruli stage. The transition from the S-shaped body

to the capillary loop stage is crucial for the differentiation of

podocytes, which are derived from mesenchymal cells [15]. During

the early renal developmental stages, immature podocyte precursor

cells exist as simple, undifferentiated epithelial cells that are

equipped with apically located tight junctions [80]. As podocytes

enter the capillary loop stage, they lose their mitotic activity and

begin to establish their characteristic complex cell architecture,

including the appearance of FPs and the reorganization of cell–cell

junctions, leading to the appearance of the SD [80], a modified

adherens junction [20]. The phenotypic conversion occurring during

the S-shaped body stage is accompanied by the expression of

Synpo and the reappearance of vimentin, a phenotypic marker of

mesenchymal cells [15].

Table 1. Comparison of SD and adherens junction (AJ)composition

SD AJ

Transmembrane

adhesion proteins

P-cadherin E-, N-, P- and

VE-cadherins

(cell type

specific)

FAT

Nephrin

Neph1–3

JAM4

Adaptor proteins a-, b- and g-

catenins

a-, b-, g- and

d–catenins

p120-catenin

Synpo

a-Actinin a-Actinin

Vinculin Vinculin

Talin Talin

Cytoskeletal

proteins

Actin Actin

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specialization of the submembranous actin cytoskeleton,the postsynaptic density (PSD) of neurons [21]. In the PSD,multiple neurotransmitter receptors and ion channels arephysically linked to a gigantic protein network through avariety of adaptor proteins, which in turn are connected

Figure 2. A simplified model for the cytoskeletal organization in podocyte

processes. MPs contain microtubules and intermediate filaments, whereas

neighboring FPs are connected by a contractile apparatus (red) consisting of

F-actin, myosin II, a-actinin-4 (A) and Synpo (S). The linkage to the GBM by adaptor

proteins and a-(a) and b- (b) integrin heterodimers is described in detail in Figure 4.

Modified, with permission, from Ref. [16].

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to actin filaments and their associated proteins [22].Signal-dependent changes in postsynaptic actin cytoske-leton dynamics are important for neuronal plasticity, thebasis of memory and learning [23]. Similarly, at the SD,multiple membrane proteins are present that are con-nected to actin through a variety of adaptor and effectorproteins (Figure 3c). The similarity between the SD andthe PSD is underscored by the observation that densin,originally described as a brain-specific synaptic protein[24], is also found at the SD, where it interacts witha–actinin-4 and nephrin [25].

The biochemical complexity of the SD protein networkmight ensure the maintenance of the SD filter structure atany cost. Indeed, the dysregulation of the SD or its loss is acommon theme in many renal diseases [26]. Alternatively,the multiprotein complex assembled at the SD mightfunction as a key sensor and regulator of the permanentchanges in FP shape and length. These changes in podo-cyte FP plasticity need to be precisely coordinatedwith FPsof neighboring podocytes so as not to compromise theintegrity of the filtration barrier during FP movements,with functional coupling of opposing FPs and signalingcascades on both sides of the SD. Therefore, the molecularcomplexity of the SD network most likely reflects a SDfunction that is far more complex than simply serving as aphysical sieve.

The ever-growing nephrin signaling nodeThe SD represents a signaling platform that contributes tothe regulation of podocyte function in health and disease[27], and, similarly to other cell–cell junctions, it is con-nected to the actin cytoskeleton, which has a pivotal part inthis regulation. An ideal candidate for a postulated trans-membrane protein that can serve as a surface receptortransmitting extracellular signals from the SD to the actincytoskeleton is nephrin. Mutations in the nephrin geneNPHS1 have been identified as the cause of congenitalnephrotic syndrome of the Finnish type [3], and reductionof nephrin expression was found in many human andexperimental proteinuric kidney diseases [28]. Nephrinis a member of the Ig superfamily, and, through its extra-cellular IgG domains, canmediate cis and trans homophilicinteractions with other nephrin molecules [29,30].Through its cytoplasmic tail, nephrin interacts withadaptor proteins that form a signaling platform around

d function by components of the actin cytoskeleton, Trends Cell Biol. (2007),

Figure 3. Protein complexes of the SD domain. (a) The contractile actin bundles in the center of the FPs are encircled by black lines, and the electron dense material at the

cytoplasmic insertion site of the SD is indicated by arrows. (b) Podocyte FPs are defined by three membrane domains: the AMD (red), the BMD (blue) and the SD (black). All

three domains are connected to the underpinning actin cytoskeleton (gray) and also with each other. Abbreviation: CA, contractile apparatus. (c) A schematic diagram of the

SD multiprotein complex and its linkage to the FP actin cytoskeleton. Membrane proteins are shown in red, adaptor proteins in orange and effector proteins in green.

Biochemical protein–protein interactions are shown by blue double arrows, and effector pathways are indicated by green arrows. Only a subset of interactions is shown.

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nephrin (Figure 3c). Among the constantly increasingnumber of nephrin-binding proteins are several transmem-brane proteins, which stabilize nephrin at the SD andfacilitate nephrin signaling [27]. The SD is organized inlipid rafts [31], and podocin, a hairpin-like raft-associatedmembrane protein, interacts directly with nephrin in theserafts [32]. Podocin also interacts with Neph1–3 [33], trans-membrane proteins of the IgG superfamily, whose presenceis also crucial for the integrity of the SD [32,34,35]. Nephproteins can homodimerize with each other but also formheterodimers with nephrin, thereby supporting nephrinstability, and SD localization and function [36].

Linking nephrin to actin bundling and nucleation

In addition to functioning as a physical link to the actincytoskeleton, nephrin also functionally connects the SD to

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modulators of actin dynamics. CD2-associated protein(CD2AP) is a cytoplasmic adaptor protein which bindsdirectly to nephrin and podocin [32,37]. It was originallyidentified as adaptor between adhesion receptors and thecytoskeleton of T cells [38]; however, it has more recentlybeen shown to have an essential role in podocytes. CD2APnull mice die of renal failure after six to seven weeks [39],and podocyte-specific expression of CD2AP rescues leth-ality of CD2AP deficiency [40]. CD2AP interacts with actin[41], the actin-binding proteins CapZ [42], cortactin [43]and the a-actinin-modulating protein Synpo (see later)[13,44] (Figure 3c). This provides a signaling cascade fromthe SD-located nephrin–CD2AP complex through Synpo tothe actin bundling protein a-actinin [13]. Furthermore,nephrin–CD2AP signaling through cortactin can also effectactin nucleation by the Arp2/3 complex [45]. This places

d function by components of the actin cytoskeleton, Trends Cell Biol. (2007),

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a-actinin and Arp2/3 as major regulators of the actincytoskeleton within the SD signaling cascade (Figure 3c).Nephrin also interacts directly with a-actinin-4 [46] and thea-actinin-binding proteins MAGUK with inverted domainstructure (MAGI)-1 [47] and MAGI-2 [46], adaptor mol-ecules that also bind to Synpo [48]. MAGI-1 and -2 cantherefore serve as a scaffold for nephrin, Synpo anda-actinin-4. Additionally, MAGI-1 links the actin cytoske-leton to junctional adhesion molecule-4 (JAM4), anotherSD membrane protein [47], but the functional significanceof this interaction is unknown. ZO-1 is a tight and adherensjunctionprotein of theMAGUKfamily and is localizedat theSD[49],where it linksmembraneproteins of theSDcomplexto the actin cytoskeleton (Figure 3c). ZO-1 interacts withNeph1–3 [50] and also binds to cortactin [51]. Proteinsof the Neph1–3 family seem to serve as adaptor proteinsbetween the nephrin transmembrane complex and the actincytoskeleton (Figure 3c).

The nephrin–Nck–N-WASP connection

Another interesting signaling pathway of recent focus thatfunctionally links nephrin with the actin cytoskeletonincludes the adaptor protein Nck [11,12]. The cytoplasmicdomain of nephrin contains six conserved tyrosine resi-dues, which can be phosphorylated by members of the

Figure 4. Schematic diagram of the BMD protein complex and its linkage to the FP actin

effector proteins in green. Biochemical protein–protein interactions are shown by blue do

interactions is shown. In addition to a3b1 integrin, the GBM domain also contains a- a

actin filaments are linked to the extracellular matrix through dystroglycan and its adap

dystroglycan and utrophin, a dystrophin analog. Dystroglycans might be responsible fo

whereas integrins serve a more regulatory function in cell–matrix adhesion and FP pla

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Src-kinase family during renal development and underpathological conditions [12,31]. Following nephrin phos-phorylation through Fyn [52], the SH2 domain ofNck bindsto phospho-nephrin, and the SH3 domains of Nck bind toneuronal Wiskott–Aldrich syndrome protein (N-WASP)[11,12]. N-WASP, in turn, activates the Arp2/3 complex,thereby linking the nephrin–Nck complex to the regulationof podocyte actin dynamics (Figure 3c). However, the phys-iological relevance of nephrin–Nck-mediated Arp2/3 acti-vation remains to be established.

SynpoSynpo is the founding member of a unique class ofproline-rich, actin-associated proteins that are expressedin highly dynamic cell compartments, such as the dendriticspine apparatus of neurons and podocyte FPs [53]. Synpoexists in three isoforms: neuronal Synpo-short, renalSynpo-long and Synpo-T, which is not expressed in thekidney of wild-type mice but is induced in podocytes ofknockout mice lacking Synpo-short and Synpo-long [13].All three isoforms bind to a-actinin and regulate the actin-bundling activity of a-actinin [13]. Synpo also interactsdirectly with CD2AP [44] and MAGI-1 [48], two adaptorproteins that link cell surface receptors to the actin cytos-keleton at the SD and theGBM, respectively (Figures 3c,4).

cytoskeleton. Membrane proteins are shown in red, adaptor proteins in orange and

uble arrows, and effector pathways are indicated by green arrows. Only a subset of

nd b-dystroglycan as adhesion receptors [17]. Similarly to muscle fibers, in which

tor protein dystrophin, the GBM is connected with the actin cytoskeleton through

r the stable physical interaction between the sole plate and the extracellular matrix,

sticity [17]. FAK, focal adhesion kinase.

d function by components of the actin cytoskeleton, Trends Cell Biol. (2007),

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Hence, Synpo can be described as a scaffolding proteinconnecting the signaling complexes of the SD and the BMDby virtue of binding to a-actinin and the actin cytoskeleton.Of note, the physical linkage to existing actin filaments isnot the only function of Synpo. In the absence of all threeSynpo isoforms, podocytes lack stress fibers [13] and showimpaired cell migration [14], indicating an essential role ofSynpo in the regulation of podocyte actin dynamics ratherthan simply functioning by stabilizing existing actin fibers.Indeed, when the underlying molecular mechanism ofstress fiber formation in podocytes was analyzed in detail,RhoA, a major player in stress fiber formation [54], wasidentified as a novel target of Synpo [14]. It is well knownthat the activity of RhoA – and other small G proteins – canbe regulated by nucleotide loading [54] (Figure 5a). In theGTP-bound form, Rho GTPases acquire an active confor-mation, which in the case of RhoA induces stress fiberformation, whereas GDP binding results in the inacti-vation of the G protein [54].The switch in nucleotide load-ing and G protein activity are assisted by guaninenucleotide exchange factors (GEFs) andGTPase-activating

Figure 5. Smurf-1, Synpo and the proteasomal regulation of RhoA signaling. (a) Canon

GDP-bound and an active GTP-bound form. The cycle is tightly regulated mainly by GEF

such as kinases and scaffold proteins [54]. (b) More recent studies show that the E

ubiquitination, leading to its proteasomal degradation [55,56]. Synpo induces stress fib

preventing the targeting of RhoA for proteasomal degradation [14]. (c) Schematic dia

Compared with the BMD and the SD, much less is known about protein complexes linkin

well-developed glycocalyx, which contributes to the negative charge of the filtration barr

other [19]. Podocalyxin is linked to the FP actin cytoskeleton through the adaptor protei

disruption of the podocalyxin complex results in changes in the FP structure [19], and

involved in actin organization and FP formation. Another transmembrane protein of the

[79]. However, ligands or substrates for GLEPP1 remain to be identified. Abbreviation:

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proteins (GAPs) [54]. GEFs promote the exchange of GDPby GTP and thereby G protein activation, whereas GAPspromote GTP hydrolysis and inactivation of the G protein[54] (Figure 5a). For a long time, GEFs and GAPs wereconsidered to be the only regulators of RhoA. However,recent studies showed that RhoA can also be controlledby Smurf-1-mediated proteasomal degradation [55,56](Figure 5b). Smurf-1 is a HECT domain E3 ubiquitin ligasethat promotes the degradation of GTP-loaded RhoA at theleading edge of a tumor cell, thereby preventing stress fiberformation at the migration front [55,56]. Subsequent workidentified Synpo as the first inhibitor of Smurf-1-regulateddegradation of RhoA (Figure 5b). Mechanistically, Synpocompetes with Smurf-1 for RhoA binding, thereby protect-ing RhoA from Smurf-1-mediated ubiquitination andsubsequent proteasomic degradation [14]. Moreover, thesestudies also showed that – at least in podocytes – Smurf-1not only suppresses stress fiber formation at the leadingedge, but also deeper in the cell body and thereforefunctions as a global suppressor of stress fiber formation[14].

ical model of the Rho GTPase cycle [54]. Rho GTPases switch between an inactive

s, GAPs and GDIs. In their active form, Rho GTPases can bind to effector molecules

3 ubiquitin ligase Smurf-1 binds to nucleotide-free RhoA and promotes RhoA

ers by competitive blockade of Smurf-1-mediated ubiquitination of RhoA, thereby

gram of AMD protein complexes and their linkage to the FP actin cytoskeleton.

g the AMD to the FP actin cytoskeleton. The apical surface of FPs is equipped with a

ier and preserves the podocyte cytoarchitecture by keeping the FPs apart from each

ns Na–H exchanger regulatory factor (NHERF)-1 and NHERF-2, and ezrin [9,77]. The

podocalyxin null mice fail to form FPs [78], showing that podocalyxin is crucially

AMD is glomerular epithelial protein-1 (GLEPP1), a receptor tyrosine phosphatase

BS, Bowman’s space.

d function by components of the actin cytoskeleton, Trends Cell Biol. (2007),

Box 2. ILK: a key mediator of podocyte–ECM interactions

ILK, which binds to the cytoplasmic tail of b1 integrin, has emerged

as a key mediator of podocyte–matrix interactions, and the

induction of ILK expression seems to be a common theme in

progressive podocyte damage [69]. The podocyte-specific deletion

of the ILK gene in mice caused progressive focal segmental

glomerulosclerosis, leading to renal failure [81,82], implying that

ILK has a major role in podocyte function and that the tight

regulation of ILK expression levels is crucial for a proper filtration

barrier. Using the PAN-induced podocyte injury model, it was

shown that the underlying ILK-dependent signaling mechanism

involves not only podocyte–GBM interactions, but also SD proteins

[83]. Activation of ILK in podocytes induces Wnt signaling, which in

turn leads to a reduction in CD2AP and P-cadherin expression,

podocyte detachment and activation of proliferation [83]. The

overexpression of ILK causes the rearrangement of the podocyte

actin cytoskeleton, presumably through ILK-mediated phosphoryla-

tion of a-actinin [69]. Collectively, these data make ILK a promising

candidate to function as a major signal mediator between cell–

matrix contacts and the actin cytoskeleton in podocytes. ILK also

functions as an adaptor protein that interacts with numerous

cytoskeletal proteins, such as PINCH and a-parvin, which have a

crucial role in podocyte adhesion, morphology and survival [76]. It

was also shown that ILK exists in a complex with nephrin and

a-actinin in vivo and that the selective ablation of ILK in podocytes

causes a redistribution of both proteins, whereas their overall

expression levels are not changed [82]. Hence, ILK seems to function

as an adaptor that biochemically and functionally connects the BMD

and SD signaling complexes.

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Calcium signaling at the SD through TRPC6Another interesting transmembrane protein that is linkedto the SD complex through direct binding to nephrin andpodocin is TRPC6 [7], a member of the transient receptorpotential (TRP) superfamily of cation-selective ion chan-nels and a target of hereditary human focal segmentalglomerulosclerosis, leading to kidney failure [6,7]. Ca2+

entry through the multimeric TRPC complexes has beenreported to increase cytoplasmic Ca2+ levels, causing theactivation of signaling cascades and the regulation of actindynamics [57,58]. Therefore, Ca2+ might function locally atthe SD as a second messenger whose local concentrationincrease is achieved by the transient opening of TRPC6.TRPC6 might be activated in response to environmentalstimuli which function through nephrin or other trans-membrane receptors linked to the SD. As shown in othercell types, TRPC activity can be regulated by G-protein-coupled receptors and receptor tyrosine kinases [59,60]. Itwill be interesting to see whether future studies canidentify members of both receptor families as componentsof the SD signaling complex. The detection of changes inCa2+ levels at the SD would also require the presence ofcalcium-binding proteins such as calmodulin and calmo-dulin-binding proteins. So far, only Ca2+–calmodulin-de-pendent protein kinase II (CaMKII), which is linked to theSD through interactions with densin and a-actinin-4 [61],has been described as a SD protein serving such a function.Hence, it seems possible that following activation by Ca2+,CaMKII could phosphorylate yet-to-be-identified down-stream targets that cause the recently described TPRC6-induced changes in the podocyte actin cytoskeleton [58].

Classical cadherins and the SDIn keeping with the observation that the SD represents amodified adherens junction [20] (Table 1), members of thecadherin superfamily are localized at the SD. P-cadherin, aclassical member of the cadherin superfamily that isknown to mediate homophilic interactions between cells,is present at the SD and could be a good candidate tomediate cell–cell adhesion between FPs [62]. BecauseP-cadherin null mice are viable [63], P-cadherin functionwas considered by some investigators not to be crucial forthe integrity of the filtration barrier. At present, it is notclear whether, similarly to other organs [64], differentcadherin isoforms can rescue P-cadherin deficiency inpodocytes. In keeping with this idea, cultured podocytesexpress other cadherins (e.g. E-cadherin) [20], suggestingthat podocytes use a variety of cadherins with overlappingfunctions to provide stable cell–cell adhesion. Similarly toclassical adherens junctions, the SD also contains thecadherin adaptor proteins a-, b- and g-catenin (Table 1).Among many other functions, catenins provide a physicallink to the actin cytoskeleton through a-actinin and MAGIbinding (Figure 3c). The SD also contains FAT [65], amember of the protocadherin family. FAT has a giganticextracellular domain but, instead of mediating cell–celladhesion, FAT rather seems to function in signal trans-duction, as shown in other cell types [66]. Similarly tonephrin [11,12] and P-cadherin [62], FAT is a transmem-brane protein that regulates actin dynamics. However,instead of using Arp2/3 or a-actinin as a downstream

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effector, FAT controls actin polymerization through Mena[10],which seemstopromote thegrowthof long,unbranchedfilaments by inhibiting the capping process or by recruitingother proteins to stabilize further and organize the actinfilaments into bundles [67]. FAT function at the SD isknown to be essential because FAT null mice showmassiveFP effacement and die shortly after birth [68].

The podocyte–GBM interfaceIn addition to SD regulation of actin dynamics, cytoskeletalremodeling can also occur through other adhesion com-plexes. FPs are connected to the GBM by two different setsof cell matrix adhesion complexes: integrins and dystro-glycans. Integrins are heterodimeric transmembranereceptors that bind to their ligands in the GBM (e.g.fibronectin, laminin and type IV collagen), thereby anchor-ing the cell to the underlying matrix. Furthermore, integ-rins link the GBM to the intracellular actin cytoskeletonthrough a set of integrin- and actin-associated proteinsthat include paxillin, talin, vinculin, a-actinin and filamin(Figure 4). Integrins not only serve as physical attachmentsites for the actin filaments, but also regulate actindynamics in response to extracellular stimuli by way of‘outside-in’ signaling through focal adhesion kinase, integ-rin-linked kinase (ILK; Box 2) and the actin polymerizationcomplex Arp2/3 at the integrin adhesion site [69] to modu-late actin polymerization, cell shape and motility. Integ-rins can also alter their adhesive characteristics inresponse to cellular events, thereby providing ‘inside-out’signaling [69]. The major integrin at the podocyte soleplate is a3b1. Its physiological relevance for podocytemorphology and function was first illustrated by the induc-tion of FP effacement, podocyte detachment from the GBMand proteinuria in rodents after injection with anti-b1

d function by components of the actin cytoskeleton, Trends Cell Biol. (2007),

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integrin antibodies [70]. In keeping with this, a3 integrinknockout mice die during the neonatal period, anda3-deficient podocytes are unable to form mature footprocesses [70]. In contrast to the generally held view that,in podocytes, a3b1 integrin primarily serves as adhesionreceptors, the lack of a3 integrin does not impair cell–matrix adhesion of podocytes because a3 integrin depletionresults in increased adhesion and protects against puro-mycin aminonucleoside (PAN)-induced podocyte detach-ment instead [71]. These data suggest that, rather thanserving as primary adhesion receptor, a3b1 integrinmodu-lates podocyte cell–matrix adhesion. Such a role would besimilar to the transdominant inhibitor role of a3b1 integ-rin in keratinocytes, where it can suppress the function ofother integrins [70].

In addition to integrins, the GBM is connected with thepodocyte actin cytoskeleton through a- and b-dystroglycanand utrophin [17] (Figure 4). b-dystroglycan, in turn, islaterally associated with members of the sarcoglycanfamily of proteins that mediate interconnections to severalother proteins, including b1-integrin, and therefore linkboth cell–matrix adhesion complexes together [17]. Ofnote, defects in these adhesion molecules can lead topodocyte injury and proteinuria, showing that both typesof podocyte–matrix contacts are important to maintain FPstructure and filtration barrier function [17]. Podoplaninis another transmembrane glycoprotein that is alsoexpressed at the BMD [72]. Podoplanin can directly associ-ate with ezrin and regulate the activity of RhoA in cancercells but the physiological role of podoplanin in podocytesor elsewhere is still unknown [72].

ConclusionsThe convergence of multiple interconnected signalingpathways from the cell membrane of different FP domainson the podocyte actin cytoskeleton involves not only actin-binding proteins, such as a-actinin or cortactin or the actin-nucleation complex Arp2/3, but also Rho family GTPases.Synpo has emerged as a good candidate for the promotionof RhoA-mediated stress fiber formation at the SD or BMD.However, other podocyte proteins have been implicated inthe regulation of Rho GTPase signaling. The AMD proteinpodocalyxin (Figure 5c) can activate RhoA [9], whereasb-parvin [73] and PINCH [74–76] at the GBM (Figure 4)interact with a-Pix, Nck2 and Dock180. a-Pix and Dock180serve as GEFs for Cdc42 and Rac, respectively. Futurestudies will be required to test whether Rho signaling is amajor factor in the regulation of podocyte plasticity andwhether dysregulation of Rho GTPase signaling leads topodocyte injury and renal disease, as suggested by theobservation that Rho GDI-a knockout out mice developmassive proteinuria mimicking nephrotic syndrome, lead-ing to death as a result of renal failure [8].

In summary, the podocyte is an excellent model systemto study actin cytoskeleton dynamics in a physiologicalcontext because changes in podocyte actin dynamics trans-late directly into changes in kidney function. Future stu-dies will be required to address important unansweredquestions, including the identification of extracellularligands for nephrin and other transmembrane SD proteins,the search for downstream targets of TRPC6-mediated

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Ca2+ influx, and the identification of additional proteinkinases and phosphatases that regulate podocyte actindynamics. Further investigations will also be necessaryto dissect the crosstalk between the various signalingpathways that converge on the podocyte actin cytoskeleton.

AcknowledgementsP.M. is supported by NIH grants DA18886, DK57683, DK062472 and theGeorge M. O’Brien Kidney Center DK064236; K.A. is supported by theMochida Memorial Foundation, Naioto Foundation, Ichiro KaneharaFoundation and Uehara Memorial Foundation.

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