A nanobody targeting the F-actin capping protein CapG restrains breast cancer metastasis.

Introduction: Aberrant turnover of the actin cytoskeleton is intimately associated with cancer cell migration and invasion. Frequently however, evidence is circumstantial, and a reliable assessment of the therapeutic significance of a gene product is offset by lack of inhibitors that target biologic properties of a protein, as most conventional drugs do, instead of the corresponding gene. Proteomic studies have demonstrated overexpression of CapG, a constituent of the actin cytoskeleton, in breast cancer.

Mapping cytoskeletal protein function in cells by means of nanobodies.

Nanobodies or VHHs are single domain antigen binding fragments derived from heavy-chain antibodies naturally occurring in species of the Camelidae. Due to their ease of cloning, high solubility and intrinsic stability, they can be produced at low cost. Their small size, combined with high affinity and antigen specificity, enables recognition of a broad range of structural (undruggable) proteins and enzymes alike.

The actin-capping protein CapG localizes to microtubule-dependent organelles during the cell cycle.

Extensive cross-talk between the actin and the microtubule cytoskeletons has been reported. Especially in mitosis, processes dependent on actin- and microtubule-based structures alternate and regulate each other in a complex cascade leading to division into two daughter cells. Here, we have studied the subcellular localization of the filamentous actin-capping protein CapG.

The F-actin filament capping protein CapG is a bona fide nucleolar protein.

Actin works in concert with myosin I to regulate the transcription of ribosomal genes in the nucleolus. Recently, nucleolar actin has been shown to be active in its polymeric form raising the question how actin dynamics is regulated in the nucleolus. Here, we show that the actin capping protein CapG localizes in the nucleolus of cultured cells.

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.

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.

Mutant small heat-shock protein 27 causes axonal Charcot-Marie-Tooth disease and distal hereditary motor neuropathy.

Charcot-Marie-Tooth disease (CMT) is the most common inherited neuromuscular disease and is characterized by considerable clinical and genetic heterogeneity. We previously reported a Russian family with autosomal dominant axonal CMT and assigned the locus underlying the disease (CMT2F; OMIM 606595) to chromosome 7q11-q21 (ref. 2).

Hot-spot residue in small heat-shock protein 22 causes distal motor neuropathy.

Distal hereditary motor neuropathies are pure motor disorders of the peripheral nervous system resulting in severe atrophy and wasting of distal limb muscles. In two pedigrees with distal hereditary motor neuropathy type II linked to chromosome 12q24.3, we identified the same mutation (K141N) in small heat-shock 22-kDa protein 8 (encoded by HSPB8; also called HSP22).

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.