Formin-based control of the actin cytoskeleton during cytokinesis

Cytokinesis, the terminal event in the canonical cell cycle, physically separates daughter cells following mitosis. For cleavage to occur in many eukaryotes, a cytokinetic ring must assemble and constrict between divided genomes. Although dozens of different molecules localize to and participate within the cytokinetic ring, the core machinery comprises linear actin filaments. Accordingly, formins, which nucleate and elongate F-actin (filamentous actin) for the cytokinetic ring, are required for cytokinesis in diverse species. In the present article, we discuss specific modes of formin-based actin regulation during cell division and highlight emerging mechanisms and questions on this topic. © 2013 Biochemical Society

The fission yeast cytokinesis formin Cdc12p is a barbed end actin filament capping protein gated by profilin

Cytokinesis in most eukaryotes requires the assembly and contraction of a ring of actin filaments and myosin II. The fission yeast Schizosaccharomyces pombe requires the formin Cdc12p and profilin (Cdc3p) early in the assembly of the contractile ring. The proline-rich formin homology (FH) 1 domain binds profilin, and the FH2 domain binds actin. Expression of a construct consisting of the Cdc12 FH1 and FH2 domains complements a conditional mutant of Cdc12 at the restrictive temperature, but arrests cells at the permissive temperature. Cells overexpressing Cdc12(FH1FH2)p stop growing with excessive actin cables but no contractile rings. Like capping protein, purified Cdc12(FH1FH2)p caps the barbed end of actin filaments, preventing subunit addition and dissociation, inhibits end to end annealing of filaments, and nucleates filaments that grow exclusively from their pointed ends. The maximum yield is one filament pointed end per six formin polypeptides. Profilins that bind both actin and poly-l-proline inhibit nucleation by Cdc12(FH1FH2)p, but polymerization of monomeric actin is faster, because the filaments grow from their barbed ends at the same rate as uncapped filaments. On the other hand, Cdc12(FH1FH2)p blocks annealing even in the presence of profilin. Thus, formins are profilin-gated barbed end capping proteins with the ability to initiate actin filaments from actin monomers bound to profilin. These properties explain why contractile ring assembly requires both formin and profilin and why viability depends on the ability of profilin to bind both actin and poly-l-proline

HopW1 from <i>Pseudomonas syringae</i> Disrupts the Actin Cytoskeleton to Promote Virulence in Arabidopsis

A central mechanism of virulence of extracellular bacterial pathogens is the injection into host cells of effector proteins that modify host cellular functions. HopW1 is an effector injected by the type III secretion system that increases the growth of the plant pathogen Pseudomonas syringae on the Columbia accession of Arabidopsis. When delivered by P. syringae into plant cells, HopW1 causes a reduction in the filamentous actin (F-actin) network and the inhibition of endocytosis, a known actin-dependent process. When directly produced in plants, HopW1 forms complexes with actin, disrupts the actin cytoskeleton and inhibits endocytosis as well as the trafficking of certain proteins to vacuoles. The C-terminal region of HopW1 can reduce the length of actin filaments and therefore solubilize F-actin in vitro. Thus, HopW1 acts by disrupting the actin cytoskeleton and the cell biological processes that depend on actin, which in turn are needed for restricting P. syringae growth in Arabidopsis.</p

SIN-dependent phosphoinhibition of formin multimerization controls fission yeast cytokinesis

Many eukaryotes accomplish cell division by building and constricting a medial actomyosin-based cytokinetic ring (CR). In Schizosaccharomyces pombe, a Hippo-related signaling pathway termed the septation initiation network (SIN) controls CR formation, maintenance, and constriction. However, how the SIN regulates integral CR components was unknown. Here, we identify the essential cytokinetic formin Cdc12 as a key CR substrate of SIN kinase Sid2. Eliminating Sid2-mediated Cdc12 phosphorylation leads to persistent Cdc12 clustering, which prevents CR assembly in the absence of anillin-like Mid1 and causes CRs to collapse when cytokinesis is delayed. Molecularly, Sid2 phosphorylation of Cdc12 abrogates multimerization of a previously unrecognized Cdc12 domain that confers F-actin bundling activity. Taken together, our findings identify a SIN-triggered oligomeric switch that modulates cytokinetic formin function, revealing a novel mechanism of actin cytoskeleton regulation during cell division. © 2013 Bohnert et al

Livestock grazing and vegetative filter strip buffer effects on runoff sediment, nitrate, and phosphorus losses

Livestock grazing in the Midwestern United States can result in significant levels of runoff sediment and nutrient losses to surface water resources. Some of these contaminants can increase stream eutrophication and are suspected of contributing to hypoxic conditions in the Gulf of Mexico. This research quantified effects of livestock grazing management practices and vegetative filter strip buffers on runoff depth and mass losses of total solids, nitrate-nitrogen (NO3-N), and ortho-phosphorus (PO4-P) under natural hydrologic conditions. Runoff data were collected from 12 rainfall events during 2001 to 2003 at an Iowa State University research farm in central Iowa, United States. Three vegetative buffers (paddock area:vegetative buffer area ratios of 1:0.2, 1:0.1, and 1:0 no buffer [control]) and three grazing management practices (continuous, rotational, and no grazing [control]) comprised nine treatment combinations (vegetative buffer ratio/grazing management practice) replicated in three 1.35 ha (3.34 ac) plot areas. The total 4.05 ha (10.02 ac) study area also included nine 0.4 ha (1.0 ac) paddocks and 27 vegetative buffer runoff collection units distributed in a randomized complete block design. The study site was established on uneven terrain with a maximum of 15% slopes and consisted of approximately 100% cool-season smooth bromegrass. Average paddock and vegetative buffer plant tiller densities estimated during the 2003 project season were approximately 62 million and 93 million tillers ha−1 (153 million and 230 million tillers ac−1), respectively. Runoff sample collection pipe leakage discovered and corrected during 2001 possibly reduced runoff depth and affected runoff contaminant mass losses data values. Consequently, 2001 runoff analysis results were limited to treatment comparisons within the 2001 season and were not compared with 2002 and 2003 data. Analysis results from 2001 showed no significant differences in average losses of runoff, total solids, NO3-N, and PO4-P among the nine vegetative buffer/grazing practice treatment combinations. Results from 2002 indicated significantly higher losses of runoff and total solids from 1:0 no buffer/rotational grazing and 1:0 no buffer/continuous grazing treatment combination plots, respectively, compared among other 2002 season treatment combinations. The 2003 results showed significantly higher runoff and total solids losses from 1:0 no buffer/no grazing treatment combination plots compared among all 2003 treatment combinations and from 1:0.1 vegetative buffer/no grazing treatment combination plots compared among all 2003 treatment combinations and with respective 2002 treatment combinations. However, the 2003 results indicated effective vegetative buffer performance with significantly lower runoff, total solids, and NO3-N losses from the larger 1:0.2 buffer area compared among the smaller 1:0.1 buffer area and 1:0 no buffer treatment combinations. The 2003 results also indicated a highly significant increase in losses of NO3-N from 1:0.1 buffer/no grazing treatment combination plots compared among other 2003 season treatment combinations and with respective 2002 treatment combinations. Overall results from this study suggest a shift from significantly higher 2002 season plot losses of continuous and rotational grazing treatment combinations to significantly higher 2003 season losses of no grazing treatment combinations. We speculate this shift to significantly higher runoff and contaminant losses from no grazing treatment combination plots during 2003 reflects the variability inherent to a complex and dynamic soil-water environment of livestock grazing areas. However, we also hypothesize the environmental conditions that largely consisted of a dense perennial cool-season grass type, high-relief landscape, and relatively high total rainfall depth may not necessarily include livestock grazing activities

Multi-monoubiquitylation controls VASP-mediated actin dynamics Icon for The Forest of Biologists

The actin cytoskeleton performs multiple cellular functions, and as such, actin polymerization must be tightly regulated. We previously demonstrated that reversible, non-degradative ubiquitylation regulates the function of the actin polymerase VASP in developing neurons. However, the underlying mechanism of how ubiquitylation impacts VASP activity was unknown. Here, we show that mimicking multi-monoubiquitylation of VASP at K240 and K286 negatively regulates VASP interactions with actin. Using in vitro biochemical assays, we demonstrate the reduced ability of multi-monoubiquitylated VASP to bind, bundle, and elongate actin filaments. However, multi-monoubiquitylated VASP maintained the ability to bind and protect barbed ends from capping protein. Finally, we demonstrate the electroporation of recombinant multi-monoubiquitylated VASP protein altered cell spreading morphology. Collectively, these results suggest a mechanism in which ubiquitylation controls VASP-mediated actin dynamics

Profilin-1 Serves as a Gatekeeper for Actin Assembly by Arp2/3-Dependent and -Independent Pathways

Cells contain multiple F-actin assembly pathways including the Arp2/3 complex, formins, and Ena/VASP, which have largely been analyzed separately. They collectively generate the bulk of F-actin from a common pool of G-actin; however, the interplay/competition between these pathways remains poorly understood. Using fibroblast lines derived from an Arpc2 conditional knockout mouse, we established matched-pair cells with and without the Arp2/3 complex. Arpc2−/− cells lack lamellipodia and migrate slower than WT cells, but have F-actin levels indistinguishable from controls. Actin assembly in Arpc2−/− cells was resistant to cytochalasin-D and was highly dependent on profilin-1 and Ena/VASP, but not formins. Profilin-1 depletion in WT cells increased F-actin and Arp2/3 complex in lamellipodia. Conversely, addition of exogenous profilin-1 inhibited Arp2/3 complex actin nucleation in vitro and in vivo. These observations suggest that antagonism of the Arp2/3 complex by profilin-1 in cells maintains actin homeostasis by balancing Arp2/3 complex-dependent and independent actin assembly pathways

A high-resolution map of human evolutionary constraint using 29 mammals.

The comparison of related genomes has emerged as a powerful lens for genome interpretation. Here we report the sequencing and comparative analysis of 29 eutherian genomes. We confirm that at least 5.5% of the human genome has undergone purifying selection, and locate constrained elements covering ∼4.2% of the genome. We use evolutionary signatures and comparisons with experimental data sets to suggest candidate functions for ∼60% of constrained bases. These elements reveal a small number of new coding exons, candidate stop codon readthrough events and over 10,000 regions of overlapping synonymous constraint within protein-coding exons. We find 220 candidate RNA structural families, and nearly a million elements overlapping potential promoter, enhancer and insulator regions. We report specific amino acid residues that have undergone positive selection, 280,000 non-coding elements exapted from mobile elements and more than 1,000 primate- and human-accelerated elements. Overlap with disease-associated variants indicates that our findings will be relevant for studies of human biology, health and disease

Genome sequence of an Australian kangaroo, Macropus eugenii, provides insight into the evolution of mammalian reproduction and development.

BACKGROUND: We present the genome sequence of the tammar wallaby, Macropus eugenii, which is a member of the kangaroo family and the first representative of the iconic hopping mammals that symbolize Australia to be sequenced. The tammar has many unusual biological characteristics, including the longest period of embryonic diapause of any mammal, extremely synchronized seasonal breeding and prolonged and sophisticated lactation within a well-defined pouch. Like other marsupials, it gives birth to highly altricial young, and has a small number of very large chromosomes, making it a valuable model for genomics, reproduction and development. RESULTS: The genome has been sequenced to 2 × coverage using Sanger sequencing, enhanced with additional next generation sequencing and the integration of extensive physical and linkage maps to build the genome assembly. We also sequenced the tammar transcriptome across many tissues and developmental time points. Our analyses of these data shed light on mammalian reproduction, development and genome evolution: there is innovation in reproductive and lactational genes, rapid evolution of germ cell genes, and incomplete, locus-specific X inactivation. We also observe novel retrotransposons and a highly rearranged major histocompatibility complex, with many class I genes located outside the complex. Novel microRNAs in the tammar HOX clusters uncover new potential mammalian HOX regulatory elements. CONCLUSIONS: Analyses of these resources enhance our understanding of marsupial gene evolution, identify marsupial-specific conserved non-coding elements and critical genes across a range of biological systems, including reproduction, development and immunity, and provide new insight into marsupial and mammalian biology and genome evolution