Adenomatous polyposis coli protein nucleates actin assembly and synergizes with the formin mDia1

The microtubule regulator APC is now shown to also regulate actin filament dynamics through its C-terminal “Basic” domain

Substance P Induces Rapid and Transient Membrane Blebbing in U373MG Cells in a p21-Activated Kinase-Dependent Manner

U373MG astrocytoma cells endogenously express the full-length neurokinin 1 receptor (NK1R). Substance P (SP), the natural ligand for NK1R, triggers rapid and transient membrane blebbing and we report that these morphological changes have different dynamics and intracellular signaling as compared to the changes that we have previously described in HEK293-NK1R cells. In both cell lines, the SP-induced morphological changes are Gq-independent, and they require the Rho, Rho-associated coiled-coil kinase (ROCK) signaling pathway. Using confocal microscopy we have demonstrated that tubulin is phosphorylated subsequent to cell stimulation with SP and that tubulin accumulates inside the blebs. Colchicine, a tubulin polymerization inhibitor, blocked SP-induced blebbing in U373MG but not in HEK293-NK1R cells. Although p21-activated kinase (PAK) is expressed in both cell lines, SP induced rapid phosphorylation of PAK in U373MG, but failed to phosphorylate PAK in HEK293-NK1R cells. The cell-permeable Rho inhibitor C3 transferase inhibited SP-induced PAK phosphorylation, but the ROCK inhibitor Y27632 had no effect on PAK phosphorylation, suggesting that Rho activates PAK in a ROCK-independent manner. Our study demonstrates that SP triggers rapid changes in cell morphology mediated by distinct intracellular signaling mechanisms in U373MG versus HEK293-NK1R cells

Arp2/3 complex interactions and actin network turnover in lamellipodia

Cell migration is initiated by lamellipodia—membrane-enclosed sheets of cytoplasm containing densely packed actin filament networks. Although the molecular details of network turnover remain obscure, recent work points towards key roles in filament nucleation for Arp2/3 complex and its activator WAVE complex. Here, we combine fluorescence recovery after photobleaching (FRAP) of different lamellipodial components with a new method of data analysis to shed light on the dynamics of actin assembly/disassembly. We show that Arp2/3 complex is incorporated into the network exclusively at the lamellipodium tip, like actin, at sites coincident with WAVE complex accumulation. Capping protein likewise showed a turnover similar to actin and Arp2/3 complex, but was confined to the tip. In contrast, cortactin—another prominent Arp2/3 complex regulator—and ADF/cofilin—previously implicated in driving both filament nucleation and disassembly—were rapidly exchanged throughout the lamellipodium. These results suggest that Arp2/3- and WAVE complex-driven actin filament nucleation at the lamellipodium tip is uncoupled from the activities of both cortactin and cofilin. Network turnover is additionally regulated by the spatially segregated activities of capping protein at the tip and cofilin throughout the mesh

Regulation of Fission Yeast Morphogenesis by PP2A Activator pta2

Cell polarization is key for the function of most eukaryotic cells, and regulates cell shape, migration and tissue architecture. Fission yeast, Schizosaccharomyces pombe cells are cylindrical and polarize cell growth to one or both cell tips dependent on the cell cycle stage. Whereas microtubule cytoskeleton contributes to the positioning of the growth sites by delivering polarity factors to the cell ends, the Cdc42 GTPase polarizes secretion via actin-dependent delivery and tethering of secretory vesicles to plasma membrane. How growth is restricted to cell tips and how re-initiation of tip growth is regulated in the cell cycle remains poorly understood. In this work we investigated the function of protein phosphatase type 2A (PP2A) in S. pombe morphogenesis by deleting the evolutionary conserved PTPA-type regulatory subunit that we named pta2. pta2-deleted cells showed morphological defects and altered growth pattern. Consistent with this, actin patches and active Cdc42 were mislocalized in the pta2 deletion. These defects were additive to the lack of Cdc42-GAP Rga4. pta2Δ cells show upregulated Cdc42 activity and pta2 interacts genetically with polarisome components Tea1, Tea4 and For3 leading to complete loss of cell polarity and rounded morphology. Thus, regulation of polarity by PP2A requires the polarisome and involves Pta2-dependent control of Cdc42 activity

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference

XMAP215 polymerase activity is built by combining multiple tubulin-binding TOG domains and a basic lattice-binding region

XMAP215/Dis1 family proteins positively regulate microtubule growth. Repeats at their N termini, called TOG domains, are important for this function. While TOG domains directly bind tubulin dimers, it is unclear how this interaction translates to polymerase activity. Understanding the functional roles of TOG domains is further complicated by the fact that the number of these domains present in the proteins of different species varies. Here, we take advantage of a recent crystal structure of the third TOG domain from Caenorhabditis elegans, Zyg9, and mutate key residues in each TOG domain of XMAP215 that are predicted to be important for interaction with the tubulin heterodimer. We determined the contributions of the individual TOG domains to microtubule growth. We show that the TOG domains are absolutely required to bind free tubulin and that the domains differentially contribute to XMAP215’s overall affinity for free tubulin. The mutants’ overall affinity for free tubulin correlates well with polymerase activity. Furthermore, we demonstrate that an additional basic region is important for targeting to the microtubule lattice and is critical for XMAP215 to function at physiological concentrations. Using this information, we have engineered a “bonsai” protein, with two TOG domains and a basic region, that has almost full polymerase activity

Rapid formin-mediated actin-filament elongation is essential for polarized plant cell growth

Formins are present in all eukaryotes and are essential for the creation of actin-based structures responsible for diverse cellular processes. Because multicellular organisms contain large formin gene families, establishing the physiological functions of formin isoforms has been difficult. Using RNAi, we analyzed the function of all 9 formin genes within the moss Physcomitrella patens. We show that plants lacking class II formins (For2) are severely stunted and composed of spherical cells with disrupted actin organization. In contrast, silencing of all other formins results in normal elongated cell morphology and actin organization. Consistent with a role in polarized growth, For2 are apically localized in growing cells. We show that an N-terminal phosphatase tensin (PTEN)-like domain mediates apical localization. The PTEN-like domain is followed by a conserved formin homology (FH)1-FH2 domain, known to promote actin polymerization. To determine whether apical localization of any FH1-FH2 domain mediates polarized growth, we performed domain swapping. We found that only the class II FH1-FH2, in combination with the PTEN-like domain, rescues polarized growth, because it cannot be replaced with a similar domain from a For1. We used in vitro polymerization assays to dissect the functional differences between these FH1-FH2 domains. We found that both the FH1 and the FH2 domains from For2 are required to mediate exceptionally rapid rates of actin filament elongation, much faster than any other known formin. Thus, our data demonstrate that rapid rates of actin elongation are critical for driving the formation of apical filamentous actin necessary for polarized growth