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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

YSK1 is activated by the Golgi matrix protein GM130 and plays a role in cell migration through its substrate 14-3-3zeta.

The Golgi apparatus has long been suggested to be important for directing secretion to specific sites on the plasma membrane in response to extracellular signaling events. However, the mechanisms by which signaling events are coordinated with Golgi apparatus function remain poorly understood. Here, we identify a scaffolding function for the Golgi matrix protein GM130 that sheds light on how such signaling events may be regulated.

Beta-casein-derived peptides, produced by bacteria, stimulate cancer cell invasion and motility.

In colon cancer, enteric bacteria and dietary factors are major determinants of the microenvironment but their effect on cellular invasion is not known. We therefore incubated human HCT-8/E11 colon cancer cells with bacteria or bacterial conditioned medium on top of collagen type I gels. Listeria monocytogenes stimulate cellular invasion through the formation of a soluble motility-promoting factor, identified as a 13mer beta-casein-derived peptide (HKEMPFPKYPVEP).

A kelch beta propeller featuring as a G beta structural mimic: reinventing the wheel?

New genetic and protein interaction data suggest that G protein alpha subunits may have partners with primary sequences that are quite divergent. How this is achieved may be through the adoption of similar structures, the beta propeller, by both proteins containing WD-40 repeats and kelch domains. Gettemans et al.

The Nucleo-cytoplasmic actin-binding protein CapG lacks a nuclear export sequence present in structurally related proteins.

Despite thorough structure-function analyses, it remains unclear how CapG, a ubiquitous F-actin barbed end capping protein that controls actin microfilament turnover in cells, is able to reside in the nucleus and cytoplasm, whereas structurally related actin-binding proteins are predominantly cytoplasmic. Here we report the molecular basis for the different subcellular localization of CapG, severin, and fragminP. Green fluorescent protein-tagged fragminP and severin accumulate in the nucleus upon treatment of transfected cells with the CRM1 inhibitor leptomycin B.

Gelsolin-induced epithelial cell invasion is dependent on Ras-Rac signaling.

Gelsolin is a widely distributed actin binding protein involved in controlling cell morphology, motility, signaling and apoptosis. The role of gelsolin in tumor progression, however, remains poorly understood. Here we show that expression of green fluorescent protein (GFP)-tagged gelsolin in MDCK-AZ, MDCKtsSrc or HEK293T cells promotes invasion into collagen type I.

Fragmin60 encodes an actin-binding protein with a C2 domain and controls actin Thr-203 phosphorylation in Physarum plasmodia and sclerotia.

We report the isolation of a cDNA clone encoding a 60-kDa protein termed fragmin60 that cross-reacts with fragmin antibodies. Unlike other gelsolin-related proteins, fragmin60 contains a unique N-terminal domain that shows similarity with C2 domains of aczonin, protein kinase C, and synaptotagmins. The fragmin60 C2 domain binds three calcium ions, one with nanomolar affinity and two with micromolar affinity.

Selected BTB/POZ-kelch proteins bind ATP.

Proteins with a bric-à-brac, tramtrack, broad-complex/Poxvirus zinc fingers (BTB/POZ) domain are implicated in a broad variety of biological processes, including DNA binding, regulation of gene transcription and organization of macromolecular structures. Kelch domain containing BTB/POZ proteins like Mayven and Keap1 display limited sequence similarity with the actin-fragmin kinase from Physarum, a protein kinase with a kelch domain. We show that mouse Keap1, a Caenorhabditis elegans protein that we named CKR, and human Mayven bind 5'-p-fluorosulfonyl-benzoyl-adenosine (FSBA), a covalently modifying ATP analogue.