Cytoskeleton Research Paper

The ability of cancer cells to move requires force generation to overcome factors that oppose movement (e.g. cell–cell and cell–matrix adhesions, drag, etc.). F-actin assembles with myosin II filaments composed of heavy and regulatory light chains to form a protein complex that uses energy from ATP hydrolysis to power actin–myosin contraction [32, 42]. The resultant generation of contractile force drives the morphological reorganization and extracellular matrix remodelling that facilitate cell movement. Given the profound effects that actin–myosin contractility can have, it is not surprising that there is a sophisticated network of regulatory components that hold a tight rein over this process.

Phosphorylation of the myosin II light chains (MLC) is a key mechanism for regulation of actin–myosin contractility [43]. MLC phosphorylation promotes the release of the myosin heavy chain tail allowing for assembly into filaments, and facilitates the association of the myosin head with F-actin. The myosin head uses ATP to ‘walk’ towards the barbed end. When multimeric myosin is associated with more than one actin filament this causes the filaments to move relative to each another, thereby generating contractile force. MLC phosphorylation has been reported to be mediated by numerous kinases including: the Rho-regulated ROCK1 and ROCK2 [44], the ROCK-regulated ZIPK [45], MRCKα and MRCKβ [46, 47], ILK [48], DAPK 1 [49] and 2 [50], DRAK 1 and 2 [51], PAK [52, 53] and MLCK [54] (Table 1). The ability of these various kinases to phosphorylate MLC allows for multiple signalling pathways to converge on the regulation of actin–myosin contractility. Although it would be difficult to define every condition and cell type in which a specific kinase phosphorylates MLC, studies with small molecule inhibitors indicate that ROCK1 and ROCK2 are the major calcium-independent kinases while MLCK is the major calcium-dependent kinase.

Dephosphorylation of MLC is catalyzed by the PP1M phosphatase complex, which is comprised of a PP1Cδ catalytic subunit, a myosin light chain binding subunit (MBS) and a smaller M20 subunit of unknown function [55]. The MBS is a critical component of the complex as it brings together the phosphatase catalytic subunit with its cognate substrate and because of the role it plays in regulating phosphatase activity. An interesting recent development is the discovery that there are five proteins that may act as the MBS (MYPT1, MYPT2, MYPT3, MBS85 and TIMAP) [56]. The best characterized MBS is the ubiquitously-expressed MYPT1 protein, it appears that the more tissue-restricted MYPT2 likely functions and is regulated similarly [56]. The other MBS proteins have not been studied extensively and their roles in regulating MLC phosphorylation remains to be determined. The major site of MYPT1 phosphorylation is Threonine 696 (numbering relates to the human form), which inhibits phosphatase function [57], possibly by blocking the active site or by disrupting interaction of the catalytic subunit with phosphorylated substrate [58]. Kinases that have been reported to phosphorylate Thr696 include: ROCK1 and ROCK2 [57], MRCKα and MRCKβ [47, 59], ILK [60, 61], ZIPK [62] and the DMPK [63]. Phosphorylation of Threonine 853 by ROCK has also been reported to inhibit MLC dephosphorylation by decreasing MLC binding [57, 64].

MLC phosphorylation is also regulated by the CPI-17 protein [65] (Table 1), which when phosphorylated on Threonine 38 potently inhibits PP1M activity by masking the active site in the catalytic PP1Cδ subunit [66]. A number of the same kinases that phosphorylate MYPT1 have also been shown to phosphorylate CPI-17, including ROCK1 and ROCK2 [67], ZIPK [68] and ILK [69], raising the possibility that kinases which inhibit PP1M activity do so by targeting multiple regulatory proteins. The closely related proteins KEPI and PHI-1 [70, 71] also appear to inhibit PP1C activity in a phosphorylation-dependent manner, but their possible roles in regulating MLC phosphorylation have not been characterized in detail. Elevated expression of CPI-17 in several tumour cell lines has been reported, where inhibition of PP1M led to inactivation of the Merlin tumour suppressor protein and consequent oncogenic transformation [72]. An additional possibility is that elevated CPI-17 expression and/or phosphorylation would contribute to the metastatic ability of tumour cells.

A number of kinases, including ROCK, apparently have two modes for elevating MLC phosphorylation, by acting as direct MLC kinases and by inhibiting PP1M activity. There has not been a great deal of effort spent in trying to dissect the relative contribution of these two pathways to MLC phosphorylation induced by a given kinase. However, one possibility is that the major pathway for some kinases is the phosphorylation of MYPT1 and consequent inhibition of PP1M. As a result, a net gain in MLC phosphorylation would actually require less kinase activity directed towards MLC than under conditions in which PP1M was not inhibited. A manifestation of this effect is the increased calcium sensitivity of MLC phosphorylation and the consequent actin–myosin contractile response that can be induced by ROCK [73]. In this example, it would imply that Ca2+ and/or calcium-regulated kinases such as MLCK or DAPK would cooperate with ROCK to promote contractile force generation, and contribute to metastatic behaviour.

As well as a role in facilitating MLC phosphorylation, calcium may contribute to cancer cell metastasis by binding to proteins such as S100A4 [74]. There is very strong evidence from clinical and experimental studies which indicates a significant role for S100A4 overexpression in increased metastasis and poor prognosis for a wide variety of cancers including; breast, colorectal, pancreatic and renal (Table 1). Intriguingly, S100A4 has an extracellular role in promoting metastasis, possibly by inducing remodelling of the extracellular matrix and/or through interactions with a cell surface receptor, as well as an intracellular role. It has been proposed that S100A4 acts by binding to the myosin II heavy chain [75] and promotes increased directional motility by shifting the balance towards forward protrusions and away from side protrusions [76]. In addition, S100A4 may also affect actin–myosin contractility by direct binding to F-actin [77] and to the actin-binding protein tropomyosin [78].

Tropomyosins are derived from four distinct genes (α, β, γ, δ) that are transcribed and spliced into over 40 isoforms [33, 79]. Although they play key roles in the calcium-responsive contraction of striated muscle, their roles in non-muscle cells are less well defined. Different isoforms appear to have distinct biological functions, as a result the patterns of expression affect how tropomyosins might affect the actin cytoskeleton. The expression of tropomyosin isoforms is frequently altered in tumours (Table 1). Some isoforms appear to recruit myosin to actin filaments [80], and influence the activity of the myosin head ATPase and contractility [81]. Tropomyosin has also been reported to increase actin filament stiffness [82] and protect F-actin from the actions of cofilin [83] and gelsolin [84]. However, some isoforms actually reduce active myosin levels and promote the association of cofilin with actin filaments, resulting in the formation of lamellipodia [80]. To add further complexity, isoforms are sorted to different cellular compartments, and these distributions may change during development or in tumour cells. As a result, actin–myosin regulation may be affected by factors in addition to tropomyosin expression levels. Further research is necessary to determine how both isoform expression and subcellular distribution patterns contribute to tumour cell metastasis.

Mutations in inversin cause nephronophthisis type II, an autosomal recessive form of polycystic kidney disease associated with situs inversus, dilatation, and kidney cyst formation. Since cyst formation may represent a planar polarity... more

Mutations in inversin cause nephronophthisis type II, an autosomal recessive form of polycystic kidney disease associated with situs inversus, dilatation, and kidney cyst formation. Since cyst formation may represent a planar polarity defect, we investigated whether inversin plays a role in cell division. In developing nephrons from inv-/- mouse embryos we observed heterogeneity of nuclear size, increased cell membrane perimeters, cells with double cilia, and increased frequency of binuclear cells. Depletion of inversin by siRNA in cultured mammalian cells leads to an increase in bi- or multinucleated cells. While spindle assembly, contractile ring formation, or furrow ingression appears normal in the absence of inversin, mitotic cell rounding and the underlying rearrangement of the cortical actin cytoskeleton are perturbed. We find that inversin loss causes extensive filopodia formation in both interphase and mitotic cells. These cells also fail to round up in metaphase. The resultant spindle positioning defects lead to asymmetric division plane formation and cell division. In a cell motility assay, fibroblasts isolated from inv-/- mouse embryos migrate at half the speed of wild-type fibroblasts. Together these data suggest that inversin is a regulator of cortical actin required for cell rounding and spindle positioning during mitosis. Furthermore, cell division defects resulting from improper spindle position and perturbed actin organization contribute to altered nephron morphogenesis in the absence of inversin.


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