The Arp2/3 Regulatory System and Its Deregulation in Cancer.

The Arp2/3 complex is an evolutionary conserved molecular machine that generates branched actin networks. When activated, the Arp2/3 complex contributes the actin branched junction and thus cross-links the polymerizing actin filaments in a network that exerts a pushing force. The different activators initiate branched actin networks at the cytosolic surface of different cellular membranes to promote their protrusion, movement, or scission in cell migration and membrane traffic. Here we review the structure, function, and regulation of all the direct regulators of the Arp2/3 complex that induce or inhibit the initiation of a branched actin network and that controls the stability of its branched junctions. Our goal is to present recent findings concerning novel inhibitory proteins or the regulation of the actin branched junction and place these in the context of what was previously known to provide a global overview of how the Arp2/3 complex is regulated in human cells. We focus on the human set of Arp2/3 regulators to compare normal Arp2/3 regulation in untransformed cells to the deregulation of the Arp2/3 system observed in patients affected by various cancers. In many cases, these deregulations promote cancer progression and have a direct impact on patient survival.

The trimeric coiled-coil HSBP1 protein promotes WASH complex assembly at centrosomes.

The Arp2/3 complex generates branched actin networks that exert pushing forces onto different cellular membranes. WASH complexes activate Arp2/3 complexes at the surface of endosomes and thereby fission transport intermediates containing endocytosed receptors, such as α5ß1 integrins. How WASH complexes are assembled in the cell is unknown. Here, we identify the small coiled-coil protein HSBP1 as a factor that specifically promotes the assembly of a ternary complex composed of CCDC53, WASH, and FAM21 by dissociating the CCDC53 homotrimeric precursor. HSBP1 operates at the centrosome, which concentrates the building blocks. HSBP1 depletion in human cancer cell lines and in Dictyostelium amoebae phenocopies WASH depletion, suggesting a critical role of the ternary WASH complex for WASH functions. HSBP1 is required for the development of focal adhesions and of cell polarity. These defects impair the migration and invasion of tumor cells. Overexpression of HSBP1 in breast tumors is associated with increased levels of WASH complexes and with poor prognosis for patients.

Phosphorylation of Merlin by Aurora A kinase appears necessary for mitotic progression.

Although Merlin's function as a tumor suppressor and regulator of mitogenic signaling networks such as the Ras/rac, Akt, and Hippo pathways is well-documented, in mammals as well as in insects, its role during cell cycle progression remains unclear. In this study, using a combination of approaches, including FACS analysis, time-lapse imaging, immunofluorescence microscopy, and co-immunoprecipitation, we show that Ser-518 of Merlin is a substrate of the Aurora protein kinase A during mitosis and that its phosphorylation facilitates the phosphorylation of a newly discovered site, Thr-581. We found that the expression in HeLa cells of a Merlin variant that is phosphorylation-defective on both sites leads to a defect in centrosomes and mitotic spindles positioning during metaphase and delays the transition from metaphase to anaphase. We also show that the dual mitotic phosphorylation not only reduces Merlin binding to microtubules but also timely modulates ezrin interaction with the cytoskeleton. Finally, we identify several point mutants of Merlin associated with neurofibromatosis type 2 that display an aberrant phosphorylation profile along with defective α-tubulin-binding properties. Altogether, our findings of an Aurora A-mediated interaction of Merlin with α-tubulin and ezrin suggest a potential role for Merlin in cell cycle progression.

Arpin downregulation in breast cancer is associated with poor prognosis.

BACKGROUND: The Arp2/3 complex is required for cell migration and invasion. The Arp2/3 complex and its activators, such as the WAVE complex, are deregulated in diverse cancers. Here we investigate the expression of Arpin, the Arp2/3 inhibitory protein that antagonises the WAVE complex. METHODS: We used qRT-PCR and reverse phase protein arrays in a patient cohort with known clinical parameters and outcome, immunofluorescence in breast biopsy cryosections and breast cancer cell lines. RESULTS: Arpin was downregulated at the mRNA and protein levels in mammary carcinoma cells. Arpin mRNA downregulation was associated with poor metastasis-free survival (MFS) on univariate analysis (P=0.022). High expression of the NCKAP1 gene that encodes a WAVE complex subunit was also associated with poor MFS on univariate analysis (P=0.0037) and was mutually exclusive with Arpin low. Arpin low or NCKAP1 high was an independent prognosis factor on multivariate analysis (P=0.0012) and was strongly associated with poor MFS (P=0.000064). CONCLUSIONS: Loss of the Arp2/3 inhibitory protein Arpin produces a similar poor outcome in breast cancer as high expression of the NCKAP1 subunit of the Arp2/3 activatory WAVE complex.

The Arp1/11 minifilament of dynactin primes the endosomal Arp2/3 complex.

Dendritic actin networks develop from a first actin filament through branching by the Arp2/3 complex. At the surface of endosomes, the WASH complex activates the Arp2/3 complex and interacts with the capping protein for unclear reasons. Here, we show that the WASH complex interacts with dynactin and uncaps it through its FAM21 subunit. In vitro, the uncapped Arp1/11 minifilament elongates an actin filament, which then primes the WASH-induced Arp2/3 branching reaction. In dynactin-depleted cells or in cells where the WASH complex is reconstituted with a FAM21 mutant that cannot uncap dynactin, formation of branched actin at the endosomal surface is impaired. Our results reveal the importance of the WASH complex in coordinating two complexes containing actin-related proteins.

Cortical branched actin determines cell cycle progression.

The actin cytoskeleton generates and senses forces. Here we report that branched actin networks from the cell cortex depend on ARPC1B-containing Arp2/3 complexes and that they are specifically monitored by type I coronins to control cell cycle progression in mammary epithelial cells. Cortical ARPC1B-dependent branched actin networks are regulated by the RAC1/WAVE/ARPIN pathway and drive lamellipodial protrusions. Accordingly, we uncover that the duration of the G1 phase scales with migration persistence in single migrating cells. Moreover, cortical branched actin more generally determines S-phase entry by integrating soluble stimuli such as growth factors and mechanotransduction signals, ensuing from substratum rigidity or stretching of epithelial monolayers. Many tumour cells lose this dependence for cortical branched actin. But the RAC1-transformed tumour cells stop cycling upon Arp2/3 inhibition. Among all genes encoding Arp2/3 subunits, ARPC1B overexpression in tumours is associated with the poorest metastasis-free survival in breast cancer patients. Arp2/3 specificity may thus provide diagnostic and therapeutic opportunities in cancer.

Directional Collective Migration in Wound Healing Assays.

Cell migration is suppressed by confluence in a process called contact inhibition. Relieving contact inhibition upon scratching is one of the simplest ways to induce cell migration in a variety of cell types. Wound healing is probably most relevant to epithelial monolayers, because epithelial cells generally assume a barrier function, which must be restored as fast as possible by the healing process. This versatile assay, however, can also be applied to fibroblasts and to tumor cell types. Furthermore, assessing the cell response to scratch wounding requires no special equipment or reagents. It is one of the few cell migration assays, which can even be performed without videomicroscopy, since the closure of the wound can be estimated at fixed time points. Several hours after wounding, directional collective migration is easily assessed and quantified. However, cell proliferation, which is also induced by the relief of contact inhibition, is one of the confounding factors of wound healing assays that must be taken into account. A recent alternative to the scratch-induced wound is to use special inserts to seed cells into closely spaced chambers. When the insert is removed, contact inhibition is relieved, similar to the scratch-induced wound. In this chapter, we provide the protocol of the two methods and compare their advantages and disadvantages. We also provide a protocol to estimate cell proliferation upon wound healing based on the incorporation of the nucleotide analog EdU.

WASP and WAVE Team Up at the Leading Edge.

Arp2/3-dependent branched actin networks drive membrane protrusions, with WAVE being recognized as the critical Arp2/3 activator in this process. In this issue of Developmental Cell, Zhu et al. (2016) demonstrate that WASP, an Arp2/3 activator mostly involved in endocytosis, collaborates with WAVE to promote migration of neuroblasts in Caenorhabditis elegans.