A llama-derived gelsolin single-domain antibody blocks gelsolin-G-actin interaction.

RNA interference has tremendously advanced our understanding of gene function but recent reports have exposed undesirable side-effects. Recombinant Camelid single-domain antibodies (VHHs) provide an attractive means for studying protein function without affecting gene expression. We raised VHHs against gelsolin (GsnVHHs), a multifunctional actin-binding protein that controls cellular actin organization and migration. GsnVHH-induced delocalization of gelsolin to mitochondria or the nucleus in mammalian cells reveals distinct subpopulations including free gelsolin and actin-bound gelsolin complexes. GsnVHH 13 specifically recognizes Ca(2+)-activated gelsolin (K (d) approximately 10 nM) while GsnVHH 11 binds gelsolin irrespective of Ca(2+) (K (d) approximately 5 nM) but completely blocks its interaction with G-actin. Both GsnVHHs trace gelsolin in membrane ruffles of EGF-stimulated MCF-7 cells and delay cell migration without affecting F-actin severing/capping or actin nucleation activities by gelsolin. We conclude that VHHs represent a potent way of blocking structural proteins and that actin nucleation by gelsolin is more complex than previously anticipated.

Down-regulation of myopodin expression reduces invasion and motility of PC-3 prostate cancer cells.

Enhanced motility of cancer cells by remodelling of the actin cytoskeleton is crucial in the process of cancer cell invasion and metastasis. Although several studies propose a tumor suppressor role for the actin bundling protein myopodin, it was also shown previously that overexpression of mouse myopodin promotes invasion in vitro. In the present study, the role of myopodin in human cancer cell motility and invasion was explored using RNA interference with siRNA duplexes designed to down-regulate all human myopodin isoforms currently identified. We show that down-regulation of myopodin expression in human cancer cells significantly reduces the invasive properties of these cells both in collagen type I and in Matrigel. Furthermore, the motile characteristics of cancer cells are also curbed by reduced myopodin expression whereas cell-cell contacts are reinforced. These results point to a role for myopodin as tumor activator. While these findings are at variance with the suggested tumor suppressor role for myopodin, we hypothesize that the subcellular localization of the protein is involved in its suppressor or activator function in tumorigenesis.

Multiple isoforms of the tumor suppressor myopodin are simultaneously transcribed in cancer cells.

Expression of myopodin, an actin associated protein, is frequently lost in invasive prostate cancers due to partial or complete deletion of the gene. Screening of public databases reveals that two human myopodin isoforms have been proposed. Remarkably both isoforms deviate profoundly from the human or mouse isoforms examined to date. Here, we investigated expression of human myopodin. Rapid amplification of cDNA ends revealed a new myopodin transcript, hitherto unpredicted by public databases. RT-PCR analysis indicates that the new isoform (Myo2), in addition to the two predicted isoforms (Myo1 and Myo3), are transcribed in various mammalian cell lines. The three isoforms (Myo1-3) are translated into full length proteins of 1093, 1109, and 1261 amino acids, respectively, when expressed in cells. Thus, mammalian cells simultaneously express at least three myopodin isoforms with a common N-terminal PDZ domain, but a dissimilar carboxy-terminal amino acid tract. These findings shed new light on the expression of this tumor suppressor gene and necessitate closer examination of both mouse and human myopodin polypeptides currently under study.

A new role for nuclear transport factor 2 and Ran: nuclear import of CapG.

The small GTPase Ran plays a central role in nucleocytoplasmic transport. Nuclear transport of Ran itself depends on nuclear transport factor 2 (NTF2). Here, we report that NTF2 and Ran control nuclear import of the filamentous actin capping protein CapG. In digitonin-permeabilized cells, neither GTPgammaS nor the GTP hydrolysis-deficient Ran mutant RanQ69L affect transit of CapG to the nucleus in the presence of cytosol. Obstruction of nucleoporins prevents nuclear transport of CapG, and we show that CapG binds to nucleoporin62. In addition, CapG interacts with NTF2, associates with Ran and is furthermore able to bind the NTF2-Ran complex. NTF2-Ran interaction is required for CapG nuclear import. This is corroborated by a NTF2 mutant with reduced affinity for Ran and a Ran mutant that does not bind NTF2, both of which prevent CapG import. Thus, a ubiquitously expressed protein shuttles to the nucleus through direct association with NTF2 and Ran. The role of NTF2 may therefore not be solely confined to sustaining the Ran gradient in cells.

Downregulation of gelsolin family proteins counteracts cancer cell invasion in vitro.

Gelsolin and CapG are both actin binding proteins that modulate a variety of physiological processes by interacting differently with the actin cytoskeleton. Several studies suggest that overexpression of these proteins promotes invasion in vitro. In this study we explored the contribution of these proteins in human cancer cell invasion and motility. We show that down regulation of CapG or gelsolin in several types of cancer cells, including MDA-MB 231 and PC-3 cells, significantly reduces the invasive and motile properties of cells, as well as cell aggregation. These results point to a role for CapG and gelsolin as tumor activator.

Phosphorylation on Ser5 increases the F-actin-binding activity of L-plastin and promotes its targeting to sites of actin assembly in cells.

L-plastin, a malignant transformation-associated protein, is a member of a large family of actin filament cross-linkers. Here, we analysed how phosphorylation of L-plastin on Ser5 of the headpiece domain regulates its intracellular distribution and its interaction with F-actin in transfected cells and in in vitro assays. Phosphorylated wild-type L-plastin localised to the actin cytoskeleton in transfected Vero cells. Ser5Ala substitution reduced the capacity of L-plastin to localise with peripheral actin-rich membrane protrusions. Conversely, a Ser5Glu variant mimicking a constitutively phosphorylated state, accumulated in actin-rich regions and promoted the formation of F-actin microspikes in two cell lines. Similar to phosphorylated wild-type L-plastin, this variant remained associated with cellular F-actin in detergent-treated cells, whereas the Ser5Ala variant was almost completely extracted. When compared with non-phosphorylated protein, phosphorylated L-plastin and the Ser5Glu variant bound F-actin more efficiently in an in vitro assay. Importantly, expression of L-plastin elicited collagen invasion in HEK293T cells, in a manner dependent on Ser5 phosphorylation. Based on our findings, we propose that conversely to other calponin homology (CH)-domain family members, phosphorylation of L-plastin switches the protein from a low-activity to a high-activity state. Phosphorylated L-plastin might act as an integrator of signals controlling the assembly of the actin cytoskeleton and cell motility in a 3D-space.

A monopartite nuclear localization sequence regulates nuclear targeting of the actin binding protein myopodin.

Myopodin is an actin bundling protein that shuttles between nucleus and cytoplasm in response to cell stress or during differentiation. Here, we show that the myopodin sequence 58KKRRRRARK66, when tagged to either enhanced green fluorescent protein (EGFP) or to enhanced cyan fluorescent protein-CapG (ECFPCapG), is able to target these proteins to the nucleolus in HeLa or HEK293T cells. By contrast, 58KKRR61-ECFP-CapG accumulates in the nucleus. Mutation of 58KKRRRRARK66 into alanine residues blocks myopodin nuclear import and promotes formation of cytoplasmic actin filaments. A second putative nuclear localization sequence, 612KTSKKKGKK620, displays much weaker activity in a heterologous context, and appears not to be functional in the full length protein. Thus myopodin nuclear translocation is dependent on a monopartite nuclear localization sequence.

Nuclear actin-binding proteins as modulators of gene transcription.

Dynamic transformations in the organization of the cellular microfilament system are the driving force behind fundamental biological processes such as cellular motility, cytokinesis, wound healing and secretion. Eukaryotic cells express a plethora of actin-binding proteins (ABPs) allowing cells to control the organization of the actin cytoskeleton in a flexible manner. These structural proteins were, not surprisingly, originally described as (major) constituents of the cytoplasm. However, in recent years, there has been a steady flow of reports detailing not only translocation of ABPs into and out of the nucleus but also describing their role in the nuclear compartment. This review focuses on recent developments pertaining to nucleocytoplasmic transport of ABPs, including their mode of translocation and nuclear function. In particular, evidence that structurally and functionally unrelated cytoplasmic ABPs regulate transcription activation by various nuclear (steroid hormone) receptors is steadily accruing. Furthermore, the recent finding that actin is a necessary component of the RNA polymerase II-containing preinitiation complex opens up new opportunities for nuclear ABPs in gene transcription regulation.

Molecular basis for dissimilar nuclear trafficking of the actin-bundling protein isoforms T- and L-plastin.

T- and L-plastin are highly similar actin-bundling proteins implicated in the regulation of cell morphology, lamellipodium protrusion, bacterial invasion and tumor progression. We show that T-plastin localizes predominantly to the cytoplasm, whereas L-plastin distributes between nucleus and cytoplasm in HeLa or Cos cells. T-plastin shows nuclear accumulation upon incubation of cells with the CRM1 antagonist leptomycin B (LMB). We identified a Rev-like nuclear export sequence (NES) in T-plastin that is able to export an otherwise nuclear protein in an LMB-dependent manner. Deletion of the NES promotes nuclear accumulation of T-plastin. Mutation of residues L17, F21 or L26 in the T-plastin NES inhibits nuclear efflux. L-plastin harbors a less conserved NES and lacks the F21 T-plastin residue. Insertion of a Phe residue in the L-plastin NES specifically enhances its export activity. These findings explain why both isoforms exhibit specific distribution patterns in eukaryotic cells.

Increased importin-beta-dependent nuclear import of the actin modulating protein CapG promotes cell invasion.

CapG (gCap39) is a ubiquitous gelsolin-family actin modulating protein involved in cell signalling, receptor-mediated membrane ruffling, phagocytosis and motility. CapG is the only gelsolin-related actin binding protein that localizes constitutively to both nucleus and cytoplasm. Structurally related proteins like severin and fragmin are cytoplasmic because they contain a nuclear export sequence that is absent in CapG. Increased CapG expression has been reported in some cancers but a causal role for CapG in tumour development, including invasion and metastasis, has not been explored. We show that moderate expression of green fluorescent protein-tagged CapG (CapG-EGFP) in epithelial cells induces invasion into collagen type I and precultured chick heart fragments. Nuclear export sequence-tagged CapG-EGFP fails to induce invasion, whereas point mutations in the nuclear export sequence permitting nuclear re-entry restore cellular invasion. Nuclear import of CapG is energy-dependent and requires the cytosolic receptor importin beta but not importin alpha. Nuclear CapG does not possess intrinsic transactivation activity but suppresses VP16 transactivation of a luciferase reporter gene in a dose-dependent manner. Furthermore, invasion requires signalling through the Ras-phosphoinositide 3-kinase pathway and Cdc42 or RhoA, but not Rac1. We show for the first time active nuclear import of an actin binding protein, and our findings point to a role for nuclear CapG in eliciting invasion, possibly through interfering with the cellular transcription machinery.