Formins have conserved roles in cell polarity and cytokinesis directly nucleate actin filament assembly through their FH2 domain. Here, we define the active region of yeast formin Bni1 domain show that it dimerizes. Mutations disrupt dimerization abolish activity, suggesting dimers are state domains. The protects growing barbed ends filaments from vast excesses capping protein, dimer maintains a persistent association during elongation. This is not species-specific mechanism, as activities...

A critical microtubule (MT) polarization event in cell migration is the Rho/mDia-dependent stabilization of a subset MTs oriented toward direction migration. Although mDia nucleates actin filaments, it unclear whether this or separate activity underlies MT stabilization. We generated two mutants (K853A and I704A) constitutively active version mDia2 containing formin homology domains 1 2 (FH1FH2) found that they still induced stable bound to TIP proteins EB1 APC, which have also been...

Mutations in the FIC1 gene constitute molecular defect familial intrahepatic cholestasis I (Fic1 [Byler's disease]) and benign recurrent cholestasis. This report describes localization of Fic1 rat liver intestine, as well biochemical transfection studies that support its function an energy-dependent aminophospholipid translocase. Immunocytochemistry immunoblotting membrane fractions localized to canalicular, but not basolateral, plasma domain. In small was apical epithelial cells. The...

The tumor suppressor protein adenomatous polyposis coli (APC) regulates cell protrusion and migration, processes that require the coordinated regulation of actin microtubule dynamics. APC localizes in vivo to plus ends actin-rich cortical protrusions, has well-documented direct effects on However, its potential dynamics have remained elusive. Here, we show C-terminal “basic” domain (APC-B) potently nucleates formation filaments vitro stimulates assembly cells. Nucleation is achieved by a...

Complex phosphorylation-dependent signaling networks underlie the coordination of cellular growth and division. In fission yeast Schizosaccharomyces pombe, Dual specificity tyrosine-(Y)-phosphorylation regulated kinase (DYRK) family protein Pom1 regulates cell cycle progression through mitotic inducer Cdr2 controls polarity unknown targets. Here, we sought to determine phosphorylation targets activity by SILAC-based phosphoproteomics. We defined a set high-confidence that were enriched for...

Formins are conserved proteins that nucleate actin assembly and tightly associate with the fast growing barbed ends of filaments to allow insertional growth. Most organisms express multiple formins, but it has been unclear whether they have similar or distinct activities how may be regulated differentially. We isolated compared carboxyl-terminal fragments only two formins expressed in Saccharomyces cerevisiae, Bni1 Bnr1. Bnr1 was an order magnitude more potent than nucleation processive...

Adenomatous polyposis coli (APC) protein is a large tumor suppressor that truncated in most colorectal cancers. The carboxyl-terminal third of APC mediates direct interactions with microtubules and the microtubule plus-end tracking EB1. In addition, has been localized to actin-rich regions cells, but mechanism functional significance this localization have remained unclear. Here we show purified basic domain human (APC-basic) bound directly bundled actin filaments associated stress fibers...

Cyclase-associated protein (CAP), also called Srv2 in Saccharomyces cerevisiae, is a conserved actin monomer-binding that promotes cofilin-dependent turnover vitro and vivo. However, little known about the mechanism underlying this function. Here, we show S. cerevisiae CAP binds with strong preference to ADP-G-actin (K d 0.02 μM) compared ATP-G-actin 1.9 competes directly cofilin for binding ADP-G-actin. Further, blocks monomer addition specifically barbed ends of filaments, contrast...

ATP-binding cassette (ABC)-type proteins are essential for bile formation in vertebrate liver. BSEP, MDR1, MDR2, and MRP2 ABC transporters targeted to the apical (canalicular) membrane of hepatocytes where they execute ATP-dependent transport acids, drugs, amphipathic cations, phospholipids, conjugated organic anions, respectively. Changes activity abundance canalicular regulate flow; however, little is known regarding cellular that bind their trafficking. A yeast two-hybrid screen...

Working in concert, multiple actin-binding proteins regulate the dynamic turnover of actin networks. Here, we define a novel function for conserved protein twinfilin, which until now was thought to primarily as monomer-sequestering protein. We show that purified budding yeast twinfilin (Twf1) binds and severs filaments vitro at pH below 6.0 bulk kinetic fluorescence microscopy assays. Further, use total internal reflection (TIRF) demonstrate Twf1 individual real time. It has been shown...

Formins are conserved actin nucleators which promote the assembly of filaments for formation diverse structures. In fission yeast Schizosaccharomyces pombe, formin cdc12p is required specifically in actin-based contractile ring during cytokinesis. Here, using a mutational analysis cdc12p, we identify regions responsible and localization. Profilin-binding residues FH1 domain regulate processive barbed-end capping by FH2 domain. Studies photobleaching (FRAP) sensitivity to latrunculin A...

The cortical cytoskeleton mediates a range of cellular activities such as endocytosis, cell motility, and the maintenance rigidity. Traditional polymers, including actin, microtubules, septins, contribute to cytoskeleton, but additional filament systems may also exist. In yeast cells, structures called eisosomes generate specialized domains termed MCCs cluster specific proteins at sites membrane invaginations. Here we show that core eisosome protein Pil1 forms linear filaments in fission...

Formins are a conserved family of actin assembly-promoting factors with diverse biological roles, but how their activities regulated in vivo is not well understood. In Saccharomyces cerevisiae, the formins Bni1 and Bnr1 required for assembly cables polarized cell growth. Proper cable further requires Bud6. Previously it was shown that Bud6 enhances Bni1-mediated vitro, biochemical mechanism role this activity were left unclear. Here we demonstrate specifically stimulates nucleation rather...

Eisosomes are stable domains at the plasma membrane of budding yeast Saccharomyces cerevisiae and have been proposed to function in endocytosis. composed two main cytoplasmic proteins, Pil1 Lsp1, that form a scaffold around furrow-like invaginations. We show here poorly characterized eisosome protein Seg1/Ymr086w is important for biogenesis architecture. Seg1 was required efficient incorporation into eisosomes generation normal furrows. preceded during formation established platform assembly...

Cell surface area rapidly increases during mechanical and hypoosmotic stresses. Such expansion of the plasma membrane requires ‘membrane reservoirs’ that provide buffer tension, but sources this remain poorly understood. In principle, flattening invaginations buds within could additional area, as recently shown for caveolae in animal cells. Here, we used microfluidics to study rapid yeast protoplasts, which lack rigid cell wall. To survive stress, protoplasts required eisosomes,...

Protein kinases direct polarized growth by regulating the cytoskeleton in time and space could play similar roles cell division. We found that Cdc42-activated polarity kinase Pak1 colocalizes with assembling contractile actomyosin ring (CAR) remains at division site during septation. Mutations pak1 led to defects CAR assembly genetic interactions cytokinesis mutants. Through a phosphoproteomic screen, we identified novel substrates function cytokinesis. For cytokinesis, regulates...

Protein phosphorylation regulates many steps in the cell division process including cytokinesis. In fission yeast cells, anillin-like protein Mid1 sets plane and is regulated by phosphorylation. Multiple kinases act on Mid1, but no phosphatases have been shown to regulate Mid1. Here, we discovered that conserved phosphatase PP2A-B56 required for proper cytokinesis promoting levels. We find par1∆ cells lacking primary B56 subunit divide asymmetrically due assembly of misplaced cytokinetic...