scholarly journals Roles of a Fimbrin and an α-Actinin-like Protein in Fission Yeast Cell Polarization and Cytokinesis

2001 ◽  
Vol 12 (4) ◽  
pp. 1061-1077 ◽  
Author(s):  
Jian-Qiu Wu ◽  
Jürg Bähler ◽  
John R. Pringle
Keyword(s):  
Fission Yeast ◽  
Protein Localization ◽  
Normal Growth ◽  
Growth Conditions ◽  
Cell Polarization ◽  
Dependent Manner ◽  
Cortical Actin ◽  
Ring Assembly ◽  
Or Organization

Eukaryotic cells contain many actin-interacting proteins, including the α-actinins and the fimbrins, both of which have actin cross-linking activity in vitro. We report here the identification and characterization of both an α-actinin-like protein (Ain1p) and a fimbrin (Fim1p) in the fission yeast Schizosaccharomyces pombe. Ain1p localizes to the actomyosin-containing medial ring in an F-actin–dependent manner, and the Ain1p ring contracts during cytokinesis. ain1 deletion cells have no obvious defects under normal growth conditions but display severe cytokinesis defects, associated with defects in medial-ring and septum formation, under certain stress conditions. Overexpression of Ain1p also causes cytokinesis defects, and the ain1 deletion shows synthetic effects with other mutations known to affect medial-ring positioning and/or organization. Fim1p localizes both to the cortical actin patches and to the medial ring in an F-actin–dependent manner, and several lines of evidence suggest that Fim1p is involved in polarization of the actin cytoskeleton. Although a fim1deletion strain has no detectable defect in cytokinesis, overexpression of Fim1p causes a lethal cytokinesis defect associated with a failure to form the medial ring and concentrate actin patches at the cell middle. Moreover, an ain1 fim1 double mutant has a synthetical-lethal defect in medial-ring assembly and cell division. Thus, Ain1p and Fim1p appear to have an overlapping and essential function in fission yeast cytokinesis. In addition, protein-localization and mutant-phenotype data suggest that Fim1p, but not Ain1p, plays important roles in mating and in spore formation.

scholarly journals A Comprehensive Subcellular Proteomic Survey of Salmonella Grown under Phagosome-Mimicking versus Standard Laboratory Conditions

2012 ◽  
Vol 2012 ◽  
pp. 1-12 ◽  
Author(s):  
Roslyn N. Brown ◽  
James A. Sanford ◽  
Jea H. Park ◽  
Brooke L. Deatherage ◽  
Boyd L. Champion ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Protein Localization ◽  
Subcellular Location ◽  
Growth Conditions ◽  
Standard Laboratory ◽  
Response Regulators ◽  
Regulatory Systems ◽  
Protein Properties ◽  
Good Agreement

Towards developing a systems-level pathobiological understanding of Salmonella enterica, we performed a subcellular proteomic analysis of this pathogen grown under standard laboratory and phagosome-mimicking conditions in vitro. Analysis of proteins from cytoplasmic, inner membrane, periplasmic, and outer membrane fractions yielded coverage of 25% of the theoretical proteome. Confident subcellular location could be assigned to over 1000 proteins, with good agreement between experimentally observed location and predicted/known protein properties. Comparison of protein location under the different environmental conditions provided insight into dynamic protein localization and possible moonlighting (multiple function) activities. Notable examples of dynamic localization were the response regulators of two-component regulatory systems (e.g., ArcB and PhoQ). The DNA-binding protein Dps that is generally regarded as cytoplasmic was significantly enriched in the outer membrane for all growth conditions examined, suggestive of moonlighting activities. These observations imply the existence of unknown transport mechanisms and novel functions for a subset of Salmonella proteins. Overall, this work provides a catalog of experimentally verified subcellular protein locations for Salmonella and a framework for further investigations using computational modeling.

scholarly journals Tropomyosin and Myosin-II Cellular Levels Promote Actomyosin Ring Assembly in Fission Yeast

2010 ◽  
Vol 21 (6) ◽  
pp. 989-1000 ◽  
Author(s):  
Benjamin C. Stark ◽  
Thomas E. Sladewski ◽  
Luther W. Pollard ◽  
Matthew Lord
Keyword(s):  
Motor Activity ◽  
Atpase Activity ◽  
Fission Yeast ◽  
Actin Filaments ◽  
Myosin Ii ◽  
Bound State ◽  
Contractile Ring ◽  
Ring Assembly

Myosin-II (Myo2p) and tropomyosin are essential for contractile ring formation and cytokinesis in fission yeast. Here we used a combination of in vivo and in vitro approaches to understand how these proteins function at contractile rings. We find that ring assembly is delayed in Myo2p motor and tropomyosin mutants, but occurs prematurely in cells engineered to express two copies of myo2. Thus, the timing of ring assembly responds to changes in Myo2p cellular levels and motor activity, and the emergence of tropomyosin-bound actin filaments. Doubling Myo2p levels suppresses defects in ring assembly associated with a tropomyosin mutant, suggesting a role for tropomyosin in maximizing Myo2p function. Correspondingly, tropomyosin increases Myo2p actin affinity and ATPase activity and promotes Myo2p-driven actin filament gliding in motility assays. Tropomyosin achieves this by favoring the strong actin-bound state of Myo2p. This mode of regulation reflects a role for tropomyosin in specifying and stabilizing actomyosin interactions, which facilitates contractile ring assembly in the fission yeast system.

scholarly journals Antimicrobial Effects of Equine Platelet Lysate

2021 ◽  
Vol 8 ◽  
Author(s):  
Julie Gordon ◽  
Sonsiray Álvarez-Narváez ◽  
John F. Peroni
Keyword(s):  
Bacterial Growth ◽  
Antimicrobial Properties ◽  
Normal Growth ◽  
Platelet Lysate ◽  
Resistant Bacteria ◽  
Growth Media ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
E Coli

The development of antimicrobial resistant bacteria and the lack of novel antibiotic strategies to combat those bacteria is an ever-present problem in both veterinary and human medicine. The goal of this study is to evaluate platelet lysate (PL) as a biological alternative antimicrobial product. Platelet lysate is an acellular platelet-derived product rich in growth factors and cytokines that is manufactured via plateletpheresis and pooled from donor horses. In the current study, we sought to define the antimicrobial properties of PL on select gram-positive and gram-negative bacteria. Results from an end-point in vitro assay showed that PL did not support bacterial growth, and in fact significantly reduced bacterial content compared to normal growth media. An in vitro assay was then utilized to further determine the effects on bacterial growth dynamics and showed that all strains exhibited a slower growth rate and lower yield in the presence of PL. The specific effects of PL were unique for each bacterial strain: E. coli and P. aeruginosa growth was affected in a concentration-dependent manner, such that higher amounts of PL had a greater effect, while this was not true for S. aureus or E. faecalis. Furthermore, the onset of exponential growth was delayed for E. coli and P. aeruginosa in the presence of PL, which has significant clinical implications for developing a dosing schedule. In conclusion, our findings demonstrate the potential value of PL as a broad-spectrum antimicrobial that would offer an alternative to traditional antibiotics for the treatment of bacterial infection in equine species.

scholarly journals Oxidative stress-induced parkin misfolding, aggregation, and toxicity

2020 ◽  
Author(s):  
Martin Duennwald ◽  
Gary S. Shaw ◽  
Mohammad A. Esmaeili ◽  
Jane R. Rylett ◽  
Susanne Schmid ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Disulfide Bonds ◽  
Protein Misfolding ◽  
Protein Quality ◽  
Normal Growth ◽  
Growth Conditions ◽  
Cellular Protein ◽  
Loss Of Function ◽  
Underlying Mechanisms

Abstract Background: Excess oxidative stress and protein misfolding are major hallmarks of neurodegenerative disease, including Parkinson’s disease (PD). Mutations in the genes encoding the ubiquitin ligase parkin cause autosomal recessive juvenile forms of Parkinsonism by the loss of parkin function in mitochondrial homeostasis and cellular protein quality control, generally. Dysfunction of parkin might also contribute to sporadic forms of PD, yet the underlying mechanisms remain mostly unexplored. Methods: We obtained key results from studies in human PD brains, a mouse model, yeast, cultured neuronal cells, and in vitro biochemistry. Human postmortem Medial Temporal Gyrus tissue was fixed for immunohistochemistry. We performed biochemical analyses of protein lysates from human brain, mouse brain, yeast and cells to assess parkin modification by oxidative stress under normal growth conditions and more so under oxidative stress. Results: Our results reveal that oxidative stress damages parkin by inducing the formation of aberrant intra- and inter-molecular disulfide bonds, leading to parkin misfolding and inclusion formation, which is toxic to cells. We furthermore find that parkin is most severely oxidized in its active conformation. Conclusion: Collectively, our study identifies a mechanism by which protein oxidation can contribute to neurodegeneration in PD by combining loss of function with toxic gain of function mechanisms.

TheS. pombeaurora-related kinase Ark1 associates with mitotic structures in a stage dependent manner and is required for chromosome segregation

2001 ◽  
Vol 114 (24) ◽  
pp. 4371-4384 ◽  
Author(s):  
Janni Petersen ◽  
Jeannie Paris ◽  
Martin Willer ◽  
Michel Philippe ◽  
Iain M. Hagan
Keyword(s):  
Fission Yeast ◽  
Histone H3 ◽  
Central Zone ◽  
Chromosome Condensation ◽  
Yeast Cells ◽  
Aurora B ◽  
Dependent Manner ◽  
Aurora A ◽  
Spindle Formation

Metazoans contain three aurora-related kinases. Aurora A is required for spindle formation while aurora B is required for chromosome condensation and cytokinesis. Less is known about the function of aurora C. S. pombe contains a single aurora-related kinase, Ark1. Although Ark1 protein levels remained constant as cells progressed through the mitotic cell cycle, its distribution altered during mitosis and meiosis. Throughout G2 Ark1 was concentrated in one to three nuclear foci that were not associated with the spindle pole body/centromere complex. Following commitment to mitosis Ark1 associated with chromatin and was particularly concentrated at several sites including kinetochores/centromeres. Kinetochore/centromere association diminished during anaphase A, after which it was distributed along the spindle. The protein became restricted to a small central zone that transiently enlarged as the spindle extended. As in many other systems mitotic fission yeast cells exhibit a much greater degree of phosphorylation of serine 10 of histone H3 than interphase cells. A number of studies have linked this modification with chromosome condensation. Ark1 immuno-precipitates phosphorylated serine 10 of histone H3 in vitro. This activity was highest in mitotic extracts. The absence of the histone H3 phospho-serine 10 epitope from mitotic cells in which the ark1+ gene had been deleted (ark1.Δ1); the inability of these cells to resolve their chromosomes during anaphase and the co-localisation of this phospho-epitope with Ark1 early in mitosis, all suggest that Ark1 phosphorylates serine 10 of histone H3 in vivo. ark1.Δ1 cells also exhibited a reduction in kinetochore activity and a minor defect in spindle formation. Thus the enzyme activity, localisation and phenotype arising from our manipulations of this single fission yeast aurora kinase family member suggest that this single kinase is executing functions that are separately implemented by distinct aurora A and aurora B kinases in higher systems.

scholarly journals Induction of iron regulated proteins during normal growth of Neisseria meningitidis in a chemically defined medium

1994 ◽  
Vol 36 (4) ◽  
pp. 301-310 ◽  
Author(s):  
Maria Cristina de Cunto Brandileone ◽  
Rosemeire Cobo Zanella ◽  
Vera Simonsen Dias Vieira ◽  
Claudio Tavares Sacciii ◽  
Lucimar Gonçalves Milagres ◽  
...  
Keyword(s):  
Bacterial Growth ◽  
Culture Supernatant ◽  
Culture Media ◽  
Membrane Vesicles ◽  
Normal Growth ◽  
Growth Conditions ◽  
Defined Medium ◽  
Outer Membrane Vesicles ◽  
Iron Deficient

The expression of iron regulated proteins (IRPs) in vitro has been obtained in the past by adding iron chelators to the culture after bacterial growth, in the presence of an organic iron source. We have investigated aspects concerning full expression of the meningococcal IRPs during normal growth, in defined conditions using Catlin medium, Mueller Hinton and Tryptic Soy Broth (TSB). The expression of IRPs varied between different strains with respect to Ethylenediamine Di-ortho-Hidroxy-phenyl-acetic acid (EDDA) concentrations, and according to culture medium, and also between different lots of TSB. For each strain, a specific set of IRPs were expressed and higher EDDA concentrations, or addition of glucose, or use of different culture media did not resulted in a differential expression of IRPs. We were not able to grow N. meningitidis under normal growth conditions using Desferal. We looked for a good yield of outer membrane vesicles (OMVs) expressing IRPs in iron-deficient Catlin medium containing EDDA and Hemin. Culture for 32 h at 30ºC after growing for 16 h at 37ºC supported good bacterial growth. Bacterial lysis was noted after additional 24 h at 30ºC. Approximately 4 times more OMVs was recoverable from a culture supernatant after 24 h at 30ºC than from the cells after 16 h at 37ºC. The IRP were as well expressed in OMVs from culture supernatant obtained after 24 h at 30ºC as from the cells after 16 h at 37ºC.

SYK regulates B-cell migration by phosphorylation of the F-actin interacting protein SWAP-70

Blood ◽  
2011 ◽  
Vol 117 (5) ◽  
pp. 1574-1584 ◽  
Author(s):  
Glen Pearce ◽  
Tatsiana Audzevich ◽  
Rolf Jessberger
Keyword(s):  
Cell Migration ◽  
B Cells ◽  
B Cell ◽  
Cell Polarization ◽  
Actin Binding ◽  
Lymphoid Tissues ◽  
Dependent Manner ◽  
Interacting Protein

Abstract B-cell migration into and within lymphoid tissues is not only central to the humoral immune response but also for the development of malignancies and autoimmunity. We previously demonstrated that SWAP-70, an F-actin-binding, Rho GTPase-interacting protein strongly expressed in activated B cells, is necessary for normal B-cell migration in vivo. SWAP-70 regulates integrin-mediated adhesion and cell attachment. Here we show that upon B-cell activation, SWAP-70 is extensively posttranslationally modified and becomes tyrosine phosphorylated by SYK at position 517. This phosphorylation inhibits binding of SWAP-70 to F-actin. Phospho-site mutants of SWAP-70 disrupt B-cell polarization in a dominant-negative fashion in vitro and impair migration in vivo. After CXCL12 stimulation of B cells SYK becomes activated and SWAP-70 is phosphorylated in a SYK-dependent manner. Use of the highly specific SYK inhibitor BAY61-3606 showed SYK activity is necessary for normal chemotaxis and B-cell polarization in vitro and for entry of B cells into lymph nodes in vivo. These findings demonstrate a novel requirement for SYK in migration and polarization of naive recirculating B cells and show that SWAP-70 is an important target of SYK in this pathway.

scholarly journals Bni5p, a Septin-Interacting Protein, Is Required for Normal Septin Function and Cytokinesis in Saccharomyces cerevisiae

2002 ◽  
Vol 22 (19) ◽  
pp. 6906-6920 ◽  
Author(s):  
Philip R. Lee ◽  
Sukgil Song ◽  
Hyeon-Su Ro ◽  
Chong J. Park ◽  
John Lippincott ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Localization ◽  
Growth Defect ◽  
Dependent Manner ◽  
Interacting Protein ◽  
Growth Defects ◽  
Bud Emergence ◽  
Bud Neck ◽  
And Function

ABSTRACT In the budding yeast Saccharomyces cerevisiae, the Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Sep7p/Shs1p septins assemble early in the cell cycle in a ring that marks the future cytokinetic site. The septins appear to be major structural components of a set of filaments at the mother-bud neck and function as a scaffold for recruiting proteins involved in cytokinesis and other processes. We isolated a novel gene, BNI5, as a dosage suppressor of the cdc12-6 growth defect. Overexpression of BNI5 also suppressed the growth defects of cdc10-1, cdc11-6, and sep7Δ strains. Loss of BNI5 resulted in a cytokinesis defect, as evidenced by the formation of connected cells with shared cytoplasms, and deletion of BNI5 in a cdc3-6, cdc10-1, cdc11-6, cdc12-6, or sep7Δ mutant strain resulted in enhanced defects in septin localization and cytokinesis. Bni5p localizes to the mother-bud neck in a septin-dependent manner shortly after bud emergence and disappears from the neck approximately 2 to 3 min before spindle disassembly. Two-hybrid, in vitro binding, and protein-localization studies suggest that Bni5p interacts with the N-terminal domain of Cdc11p, which also appears to be sufficient for the localization of Cdc11p, its interaction with other septins, and other critical aspects of its function. Our data suggest that the Bni5p-septin interaction is important for septin ring stability and function, which is in turn critical for normal cytokinesis.

scholarly journals Pheromone-induced polarization is dependent on the Fus3p MAPK acting through the formin Bni1p

2004 ◽  
Vol 165 (1) ◽  
pp. 99-109 ◽  
Author(s):  
Dina Matheos ◽  
Metodi Metodiev ◽  
Eric Muller ◽  
David Stone ◽  
Mark D. Rose
Keyword(s):  
Cell Fusion ◽  
Cell Polarization ◽  
Yeast Cells ◽  
Cortical Actin ◽  
Cytoplasmic Microtubule ◽  
Multiple Phenotypes ◽  
Pheromone Signaling ◽  
Mutant Phenotypes

During mating, budding yeast cells reorient growth toward the highest concentration of pheromone. Bni1p, a formin homologue, is required for this polarized growth by facilitating cortical actin cable assembly. Fus3p, a pheromone-activated MAP kinase, is required for pheromone signaling and cell fusion. We show that Fus3p phosphorylates Bni1p in vitro, and phosphorylation of Bni1p in vivo during the pheromone response is dependent on Fus3p. fus3 mutants exhibited multiple phenotypes similar to bni1 mutants, including defects in actin and cell polarization, as well as Kar9p and cytoplasmic microtubule localization. Disruption of the interaction between Fus3p and the receptor-associated Gα subunit caused similar mutant phenotypes. After pheromone treatment, Bni1p-GFP and Spa2p failed to localize to the cortex of fus3 mutants, and cell wall growth became completely unpolarized. Bni1p overexpression suppressed the actin assembly, cell polarization, and cell fusion defects. These data suggest a model wherein activated Fus3p is recruited back to the cortex, where it activates Bni1p to promote polarization and cell fusion.

scholarly journals Tubulin transport in neurons.

1996 ◽  
Vol 133 (6) ◽  
pp. 1355-1366 ◽  
Author(s):  
K E Miller ◽  
H C Joshi
Keyword(s):  
Growth Conditions ◽  
Microtubule Assembly ◽  
Dependent Manner ◽  
Slow Axonal Transport ◽  
Microtubule Inhibitor ◽  
Concentration Dependent Manner ◽  
Cell Biol ◽  
History Of ◽  
Neuron Growth

A question of broad importance in cellular neurobiology has been, how is microtubule cytoskeleton of the axon organized? It is of particular interest because of the history of conflicting results concerning the form in which tubulin is transported in the axon. While many studies indicate a stationary nature of axonal microtubules, a recent series of experiments reports that microtubules are recruited into axons of neurons grown in the presence of a microtubule-inhibitor, vinblastine (Baas, P.W., and F.J. Ahmad. 1993.J. Cell Biol. 120:1427-1437: Ahmad F.J., and P.W. Baas. 1995. J. Cell Sci, 108:2761-2769; Sharp, D.J., W. Yu, and P.W. Baas. 1995. J. Cell Biol, 130:93-103; Yu, W., and P.W. Baas. 1995. J. Neurosci. 15:6827-6833.). Since vinblastine stabilizes bulk microtubule-dynamics in vitro, it was concluded that preformed microtubules moved into newly grown axons. By visualizing the polymerization of injected fluorescent tubulin, we show that substantial microtubule polymerization occurs in neurons grown at reported vinblastine concentrations. Vinblastine inhibits, in a concentration-dependent manner, both neurite outgrowth and microtubule assembly. More importantly, the neuron growth conditions of low vinblastine concentration allowed us to visualize the footprints of the tubulin wave as it polymerized and depolymerized during its slow axonal transport. In contrast, depolymerization resistant fluorescent microtubules did not move when injected in neurons. We show that tubulin subunits, not microtubules, are the primary form of tubulin transport in neurons.

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