scholarly journals Of Bars and Rings: Hof1-Dependent Cytokinesis in Multiseptated Hyphae of Ashbya gossypii

2008 ◽  
Vol 29 (3) ◽  
pp. 771-783 ◽  
Author(s):  
Andreas Kaufmann ◽  
Peter Philippsen
Keyword(s):  
Ashbya Gossypii ◽  
Actin Ring ◽  
Src Homology 3 ◽  
Filamentous Ascomycete ◽  
Multinucleated Cells ◽  
Ring Assembly ◽  
Src Homology ◽  
Type Ii Myosin ◽  
First Time

ABSTRACT We analyzed the development of multiple septa in elongated multinucleated cells (hyphae) of the filamentous ascomycete Ashbya gossypii in which septation is apparently uncoupled from nuclear cycles. A key player for this compartmentalization is the PCH protein Hof1. Hyphae that are lacking this protein form neither actin rings nor septa but still elongate at wild-type speed. Using in vivo fluorescence microscopy, we present for the first time the coordination of cytokinesis and septation in multiseptated and multinucleated cells. Hof1, the type II myosin Myo1, the landmark protein Bud3, and the IQGAP Cyk1 form collars of cortical bars already adjacent to hyphal tips, thereby marking the sites of septation. While hyphae continue to elongate, these proteins gradually form cortical rings. This bar-to-ring transition depends on Hof1 and Cyk1 but not Myo1 and is required for actin ring assembly. The Fes/CIP4 homology (FCH) domain of Hof1 ensures efficient localization of Hof1, whereas ring integrity is conferred by the Src homology 3 (SH3) domain. Up to several hours after site selection, actin ring contraction leads to membrane invagination and subsequent cytokinesis. Simultaneously, a septum forms between the adjacent hyphal compartments, which do not separate. During evolution, A. gossypii lost the homologs of two enzymes essential for cell separation in Saccharomyces cerevisiae.

scholarly journals The PI3K pathway drives the maturation of mast cells via microphthalmia transcription factor

Blood ◽  
2011 ◽  
Vol 118 (13) ◽  
pp. 3459-3469 ◽  
Author(s):  
Peilin Ma ◽  
Raghuveer Singh Mali ◽  
Veerendra Munugalavadla ◽  
Subha Krishnan ◽  
Baskar Ramdas ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Mast Cells ◽  
Pi3k Pathway ◽  
Regulatory Subunit ◽  
Growth And Survival ◽  
Src Homology 3 ◽  
Pi3k Activation ◽  
Src Homology ◽  
Microphthalmia Transcription Factor

Abstract Mast cell maturation is poorly understood. We show that enhanced PI3K activation results in accelerated maturation of mast cells by inducing the expression of microphthalmia transcription factor (Mitf). Conversely, loss of PI3K activation reduces the maturation of mast cells by inhibiting the activation of AKT, leading to reduced Mitf but enhanced Gata-2 expression and accumulation of Gr1+Mac1+ myeloid cells as opposed to mast cells. Consistently, overexpression of Mitf accelerates the maturation of mast cells, whereas Gata-2 overexpression mimics the loss of the PI3K phenotype. Expressing the full-length or the src homology 3– or BCR homology domain–deleted or shorter splice variant of the p85α regulatory subunit of PI3K or activated AKT or Mitf in p85α-deficient cells restores the maturation but not growth. Although deficiency of both SHIP and p85α rescues the maturation of SHIP−/− and p85α−/− mast cells and expression of Mitf; in vivo, mast cells are rescued in some, but not all tissues, due in part to defective KIT signaling, which is dependent on an intact src homology 3 and BCR homology domain of p85α. Thus, p85α-induced maturation, and growth and survival signals, in mast cells can be uncoupled.

scholarly journals Molecular Evolution of Podocyte Slit-diaphragm Proteins

2020 ◽  
Author(s):  
NSK Mulukala ◽  
V Kambhampati ◽  
SAH Qadri ◽  
AK Pasupulati
Keyword(s):  
Protein Complexes ◽  
Slit Diaphragm ◽  
Ankyrin Repeats ◽  
Protein Loss ◽  
Acid Base Balance ◽  
Src Homology 3 ◽  
Base Balance ◽  
Src Homology ◽  
Spfh Domain ◽  
First Time

AbstractVertebrates kidneys contribute to the homeostasis by regulating electrolyte, acid-base balance, and prevent protein loss into the urine. Glomerular podocytes constitute blood-urine barrier and podocyte slit-diaphragm, a modified tight junction contributes to the glomerular permselectivity. Nephrin, podocin, CD2AP, and TRPC6 are considered to be crucial members, which largely interact with each other and contribute to the structural and functional integrity of the slit-diaphragm. In this study, we analyzed the distribution of these four-key slit-diaphragm proteins across the organisms for which the genome sequence is available. We found that nephrin has a diverse distribution ranging from nematodes to higher vertebrates whereas podocin, CD2AP, and TRPC6 are predominantly restricted to the vertebrates. In the invertebrates nephrin and its orthologs consist of more immunoglobulin-3 and immunoglobulin-5 domains when compared to the vertebrates wherein, CD80-like C2-set Ig2 domains were predominant. Src Homology-3 (SH3) domain of CD2AP and SPFH domain of podocin are highly conserved among vertebrates. Although the majority of the TRPC6 and its orthologs had conserved ankyrin repeats, TRP, and ion transport domains, the orthologs of TRPC6 present in Rhincodon typus and Acanthaster planci do not possess the ankyrin repeats. Intrinsically unstructured regions (IURs), which are considered to contribute to the interactions among these proteins are largely conserved among orthologs of these proteins, suggesting the importance of IURs in the protein complexes that constitute slit-diaphragm. This study for the first time reports the evolutionary insights of vertebrate slit-diaphragm proteins and its invertebrate orthologs.

scholarly journals Switch-like Arp2/3 activation upon WASP and WIP recruitment to an apparent threshold level by multivalent linker proteins in vivo

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Yidi Sun ◽  
Nicole T Leong ◽  
Tommy Jiang ◽  
Astou Tangara ◽  
Xavier Darzacq ◽  
...  
Keyword(s):  
Quantitative Imaging ◽  
Threshold Level ◽  
Actin Assembly ◽  
Cellular Processes ◽  
Src Homology 3 ◽  
Filament Assembly ◽  
Tightly Coupled ◽  
Src Homology 3 Domain ◽  
Src Homology

Actin-related protein 2/3 (Arp2/3) complex activation by nucleation promoting factors (NPFs) such as WASP, plays an important role in many actin-mediated cellular processes. In yeast, Arp2/3-mediated actin filament assembly drives endocytic membrane invagination and vesicle scission. Here we used genetics and quantitative live-cell imaging to probe the mechanisms that concentrate NPFs at endocytic sites, and to investigate how NPFs regulate actin assembly onset. Our results demonstrate that SH3 (Src homology 3) domain-PRM (proline-rich motif) interactions involving multivalent linker proteins play central roles in concentrating NPFs at endocytic sites. Quantitative imaging suggested that productive actin assembly initiation is tightly coupled to accumulation of threshold levels of WASP and WIP, but not to recruitment kinetics or release of autoinhibition. These studies provide evidence that WASP and WIP play central roles in establishment of a robust multivalent SH3 domain-PRM network in vivo, giving actin assembly onset at endocytic sites a switch-like behavior.

scholarly journals The Anaphase-Promoting Complex/Cyclosome Is Required for Anaphase Progression in Multinucleated Ashbya gossypii Cells

2006 ◽  
Vol 6 (2) ◽  
pp. 182-197 ◽  
Author(s):  
Amy S. Gladfelter ◽  
Nicoleta Sustreanu ◽  
A. Katrin Hungerbuehler ◽  
Sylvia Voegeli ◽  
Virginie Galati ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Nuclear Division ◽  
Eukaryotic Cell ◽  
Ashbya Gossypii ◽  
Anaphase Promoting Complex ◽  
Cycle Progression ◽  
Filamentous Ascomycete ◽  
Multinucleated Cells ◽  
Mitotic Cyclin

ABSTRACT Regulated protein degradation is essential for eukaryotic cell cycle progression. The anaphase-promoting complex/cyclosome (APC/C) is responsible for the protein destruction required for the initiation of anaphase and the exit from mitosis, including the degradation of securin and B-type cyclins. We initiated a study of the APC/C in the multinucleated, filamentous ascomycete Ashbya gossypii to understand the mechanisms underlying the asynchronous mitosis observed in these cells. These experiments were motivated by previous work which demonstrated that the mitotic cyclin AgClb1/2p persists through anaphase, suggesting that the APC/C may not be required for the division cycle in A. gossypii. We have now found that the predicted APC/C components AgCdc23p and AgDoc1p and the targeting factors AgCdc20p and AgCdh1p are essential for growth and nuclear division. Mutants lacking any of these factors arrest as germlings with nuclei blocked in mitosis. A likely substrate of the APC/C is the securin homologue AgPds1p, which is present in all nuclei in hyphae except those in anaphase. The destruction box sequence of AgPds1p is required for this timed disappearance. To investigate how the APC/C may function to degrade AgPds1p in only the subset of anaphase nuclei, we localized components and targeting subunits of the APC/C. Remarkably, AgCdc23p, AgDoc1p, and AgCdc16p were found in all nuclei in all cell cycle stages, as were the APC/C targeting factors AgCdc20p and AgCdh1p. These data suggest that the AgAPC/C may be constitutively active across the cell cycle and that proteolysis in these multinucleated cells may be regulated at the level of substrates rather than by the APC/C itself.

scholarly journals Dephosphorylation of Iqg1 by Cdc14 regulates cytokinesis in budding yeast

2015 ◽  
Vol 26 (16) ◽  
pp. 2913-2926 ◽  
Author(s):  
Daniel P. Miller ◽  
Hana Hall ◽  
Ryan Chaparian ◽  
Madison Mara ◽  
Alison Mueller ◽  
...  
Keyword(s):  
Ring Formation ◽  
Cyclin Dependent Kinase ◽  
Filamentous Actin ◽  
Actin Ring ◽  
Ring Assembly ◽  
Cdk Phosphorylation ◽  
Necessary And Sufficient ◽  
Type Ii Myosin ◽  
Actin Rings

Cytokinesis separates cells by contraction of a ring composed of filamentous actin (F-actin) and type II myosin. Iqg1, an IQGAP family member, is an essential protein in Saccharomyces cerevisiae required for assembly and contraction of the actomyosin ring. Localization of F-actin to the ring occurs only after anaphase and is mediated by the calponin homology domain (CHD) of Iqg1, but the regulatory mechanisms that temporally restrict actin ring assembly are not well defined. We tested the hypothesis that dephosphorylation of four perfect cyclin-dependent kinase (Cdk) sites flanking the CHD promotes actin ring formation, using site-specific alanine mutants. Cells expressing the nonphosphorylatable iqg1-4A allele formed actin rings before anaphase and exhibited defects in myosin contraction and cytokinesis. The Cdc14 phosphatase is required for normal cytokinesis and acts on specific Cdk phosphorylation sites. Overexpression of Cdc14 resulted in premature actin ring assembly, whereas inhibition of Cdc14 function prevented actin ring formation. Cdc14 associated with Iqg1, dependent on several CHD-flanking Cdk sites, and efficiently dephosphorylated these sites in vitro. Of importance, the iqg1-4A mutant rescued the inability of cdc14-1 cells to form actin rings. Our data support a model in which dephosphorylation of Cdk sites around the Iqg1 CHD by Cdc14 is both necessary and sufficient to promote actin ring formation. Temporal control of actin ring assembly by Cdk and Cdc14 may help to ensure that cytokinesis onset occurs after nuclear division is complete.

scholarly journals Abelson Interactor 1 (Abi1) and Its Interaction with Wiskott-Aldrich Syndrome Protein (Wasp) Are Critical for Proper Eye Formation in Xenopus Embryos

2013 ◽  
Vol 288 (20) ◽  
pp. 14135-14146 ◽  
Author(s):  
Arvinder Singh ◽  
Emily F. Winterbottom ◽  
Yon Ju Ji ◽  
Yoo-Seok Hwang ◽  
Ira O. Daar
Keyword(s):  
Progenitor Cells ◽  
Cultured Cells ◽  
Scaffold Protein ◽  
Wiskott Aldrich Syndrome ◽  
Src Homology 3 ◽  
Eye Field ◽  
Src Homology 3 Domain ◽  
Src Homology ◽  
Syndrome Protein

Abl interactor 1 (Abi1) is a scaffold protein that plays a central role in the regulation of actin cytoskeleton dynamics as a constituent of several key protein complexes, and homozygous loss of this protein leads to embryonic lethality in mice. Because this scaffold protein has been shown in cultured cells to be a critical component of pathways controlling cell migration and actin regulation at cell-cell contacts, we were interested to investigate the in vivo role of Abi1 in morphogenesis during the development of Xenopus embryos. Using morpholino-mediated translation inhibition, we demonstrate that knockdown of Abi1 in the whole embryo, or specifically in eye field progenitor cells, leads to disruption of eye morphogenesis. Moreover, signaling through the Src homology 3 domain of Abi1 is critical for proper movement of retinal progenitor cells into the eye field and their appropriate differentiation, and this process is dependent upon an interaction with the nucleation-promoting factor Wasp (Wiskott-Aldrich syndrome protein). Collectively, our data demonstrate that the Abi1 scaffold protein is an essential regulator of cell movement processes required for normal eye development in Xenopus embryos and specifically requires an Src homology 3 domain-dependent interaction with Wasp to regulate this complex morphogenetic process.

scholarly journals The F-BAR protein Hof1 tunes formin activity to sculpt actin cables during polarized growth

2014 ◽  
Vol 25 (11) ◽  
pp. 1730-1743 ◽  
Author(s):  
Brian R. Graziano ◽  
Hoi-Ying E. Yu ◽  
Salvatore L. Alioto ◽  
Julian A. Eskin ◽  
Casey A. Ydenberg ◽  
...  
Keyword(s):  
Secretory Vesicle ◽  
Polarized Growth ◽  
Vesicle Traffic ◽  
Src Homology 3 ◽  
Polarized Secretion ◽  
Cytoskeleton Remodeling ◽  
Src Homology ◽  
Actin Cables

Asymmetric cell growth and division rely on polarized actin cytoskeleton remodeling events, the regulation of which is poorly understood. In budding yeast, formins stimulate the assembly of an organized network of actin cables that direct polarized secretion. Here we show that the Fer/Cip4 homology–Bin amphiphysin Rvs protein Hof1, which has known roles in cytokinesis, also functions during polarized growth by directly controlling the activities of the formin Bnr1. A mutant lacking the C-terminal half of Hof1 displays misoriented and architecturally altered cables, along with impaired secretory vesicle traffic. In vitro, Hof1 inhibits the actin nucleation and elongation activities of Bnr1 without displacing the formin from filament ends. These effects depend on the Src homology 3 domain of Hof1, the formin homology 1 (FH1) domain of Bnr1, and Hof1 dimerization, suggesting a mechanism by which Hof1 “restrains” the otherwise flexible FH1-FH2 apparatus. In vivo, loss of inhibition does not alter actin levels in cables but, instead, cable shape and functionality. Thus Hof1 tunes formins to sculpt the actin cable network.

scholarly journals CrfP, a fratricide protein, contributes to natural transformation in Streptococcus suis

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Yinchu Zhu ◽  
Jiale Ma ◽  
Yue Zhang ◽  
Xiaojun Zhong ◽  
Qiankun Bai ◽  
...  
Keyword(s):  
Natural Transformation ◽  
Potential Effect ◽  
Transformation Process ◽  
Transformation Rate ◽  
Bacterial Cells ◽  
Exogenous Dna ◽  
Src Homology 3 ◽  
Src Homology ◽  
Natural Genetic Transformation

AbstractStreptococcussuis (S.suis) is an important zoonotic pathogen that causes septicaemia, meningitis and streptococcal toxic shock-like syndrome in its host, and recent studies have shown that S.suis could be competent for natural genetic transformation. Transformation is an important mechanism for the horizontal transfer of DNA, but some elements that affect the transformation process need to be further explored. Upon entering the competent state, Streptococcus species stimulate the transcription of competence-related genes that are responsible for exogenous DNA binding, uptake and processing. In this study, we performed conserved promoter motif and qRT-PCR analyses and identified CrfP as a novel murein hydrolase that is widespread in S.suis and stimulated with a peptide pheromone in the competent state through a process controlled by ComX. A bioinformatics analysis revealed that CrfP consists of a CHAP hydrolase domain and two bacterial Src homology 3-binding (SH3b) domains. Further characterization showed that CrfP could be exported to extracellular bacterial cells and lytic S.suis strains of different serotypes, and this finding was verified by TEM and a turbidity assay. To investigate the potential effect of CrfP in vivo, a gene-deletion mutant (ΔcrfP) was constructed. Instead of stopping the natural transformation process, the inactivation of CrfP clearly reduced the effective transformation rate. Overall, these findings provide evidence showing that CrfP is important for S.suis serovar 2 competence.

scholarly journals Mechanism of Nuclear Movements in a Multinucleated Cell

2016 ◽  
Author(s):  
Romain Gibeaux ◽  
Antonio Z. Politi ◽  
Peter Philippsen ◽  
François Nédélec
Keyword(s):  
Pathogenic Fungus ◽  
Hyphal Growth ◽  
Stochastic Simulations ◽  
Ashbya Gossypii ◽  
Multinucleated Cell ◽  
Minimal Set ◽  
Multinucleated Cells ◽  
Nuclear Clusters ◽  
Cytoplasmic Microtubules

ABSTRACTMultinucleated cells are important in many organisms but the mechanisms governing the movements of nuclei sharing a common cytoplasm are not understood. In the hyphae of the plant pathogenic fungusAshbya gossypii,nuclei move back and forth, occasionally bypassing each other, and, preventing the formation of nuclear clusters, this is essential for genetic stability. These movements depend on cytoplasmic microtubules emanating from the nuclei, that are pulled by dynein motors anchored at the cortex. Using 3D stochastic simulations with parameters constrained by the literature, we predict the cortical anchors density from the characteristics of nuclear movements. Altogether, the model accounts for the complex nuclear movements seenin vivo,using a minimal set of experimentally determined ingredients. Interestingly, these ingredients power the oscillations of the anaphase spindle in budding yeast, but inA. gossypiithis system is not restricted to a specific nuclear cycle stage, possibly as a result of adaptation to hyphal growth and multinuclearity.

The Cdc42-associated kinase ACK1 is not autoinhibited but requires Src for activation

2011 ◽  
Vol 435 (2) ◽  
pp. 355-364 ◽  
Author(s):  
Wing Chan ◽  
Soon-Tuck Sit ◽  
Ed Manser
Keyword(s):  
Egf Receptor ◽  
Catalytic Domain ◽  
Point Mutations ◽  
Src Homology 3 ◽  
Binding Domains ◽  
Src Homology ◽  
Epidermal Growth ◽  
Tyrosine Phosphorylated Protein ◽  
External Stimuli

The non-RTK (receptor tyrosine kinase) ACK1 [activated Cdc42 (cell division cycle 42)-associated kinase 1] binds a number of RTKs and is associated with their endocytosis and turnover. Its mode of activation is not well established, but models have suggested that this is an autoinhibited kinase. Point mutations in its SH3 (Src homology 3)- or EGF (epidermal growth factor)-binding domains have been reported to activate ACK1, but we find neither of the corresponding W424K or F820A mutations do so. Indeed, deletion of the various ACK1 domains C-terminal to the catalytic domain are not associated with increased activity. A previous report identified only one major tyrosine phosphorylated protein of 60 kDa co-purified with ACK1. In a screen for new SH3 partners for ACK1 we found multiple Src family kinases; of these c-Src itself binds best. The SH2 and SH3 domains of Src interact with ACK1 Tyr518 and residues 623–652 respectively. Src targets the ACK1 activation loop Tyr284, a poor autophosphorylation site. We propose that ACK1 fails to undergo significant autophosphorylation on Tyr284in vivo because it is basophilic (whereas Src is acidophilic). Subsequent ACK1 activation downstream of receptors such as EGFR (EGF receptor) (and Src) promotes turnover of ACK1 in vivo, which is blocked by Src inhibitors, and is compromised in the Src-deficient SYF cell line. The results of the present study can explain why ACK1 is responsive to so many external stimuli including RTKs and integrin ligation, since Src kinases are commonly recruited by multiple receptor systems.

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