scholarly journals The RHO1-specific GTPase-activating Protein LRG1 Regulates Polar Tip Growth in Parallel to Ndr Kinase Signaling inNeurospora

2008 ◽  
Vol 19 (11) ◽  
pp. 4554-4569 ◽  
Author(s):  
Nico Vogt ◽  
Stephan Seiler
Keyword(s):  
Tip Growth ◽  
Rho Gtpase ◽  
Synthetic Lethality ◽  
Genetic Evidence ◽  
Active Growth ◽  
Branch Formation ◽  
Morphological Defects ◽  
Tip Extension ◽  
Ndr Kinase

Regulation of Rho GTPase signaling is critical for cell shape determination and polarity. Here, we investigated the role of LRG1, a novel member of the GTPase-activating proteins (GAPs) of Neurospora crassa. LRG1 is essential for apical tip extension and to restrict excessive branch formation in subapical regions of the hypha and is involved in determining the size of the hyphal compartments. LRG1 localizes to hyphal tips and sites of septation via its three LIM domains. The accumulation of LRG1 as an apical cap is dependent on a functional actin cytoskeleton and active growth, and is influenced by the opposing microtubule-dependent motor proteins dynein and kinesin-1. Genetic evidence and in vitro GTPase assays identify LRG1 as a RHO1-specific GAP affecting several output pathways of RHO1, based on hyposensitivity to the glucan inhibitor caspofungin, synthetic lethality with a hyperactive β1,3-glucan synthase mutant, altered PKC/MAK1 pathway activities, and hypersensitivity to latrunculin A. The morphological defects of lrg-1 are highly reminiscent to the Ndr kinase/RAM pathway mutants cot-1 and pod-6, and genetic evidence suggests that RHO1/LRG1 function in parallel with COT1 in coordinating apical tip growth.

scholarly journals The STE20/Germinal Center Kinase POD6 Interacts with the NDR Kinase COT1 and Is Involved in Polar Tip Extension inNeurospora crassa

2006 ◽  
Vol 17 (9) ◽  
pp. 4080-4092 ◽  
Author(s):  
Stephan Seiler ◽  
Nico Vogt ◽  
Carmit Ziv ◽  
Rena Gorovits ◽  
Oded Yarden
Keyword(s):  
Protein Kinase ◽  
Physical Interaction ◽  
Dependent Manner ◽  
Growth Defects ◽  
Polar Cell ◽  
Branch Formation ◽  
Tip Extension ◽  
Altered Cell ◽  
Extragenic Suppressors ◽  
Ndr Kinase

Members of the Ste20 and NDR protein kinase families are important for normal cell differentiation and morphogenesis in various organisms. We characterized POD6 (NCU02537.2), a novel member of the GCK family of Ste20 kinases that is essential for hyphal tip extension and coordinated branch formation in the filamentous fungus Neurospora crassa. pod-6 and the NDR kinase mutant cot-1 exhibit indistinguishable growth defects, characterized by cessation of cell elongation, hyperbranching, and altered cell-wall composition. We suggest that POD6 and COT1 act in the same genetic pathway, based on the fact that both pod-6 and cot-1 can be suppressed by 1) environmental stresses, 2) altering protein kinase A activity, and 3) common extragenic suppressors (ropy, as well as gul-1, which is characterized here as the ortholog of the budding and fission yeasts SSD1 and Sts5, respectively). Unlinked noncomplementation of cot-1/pod-6 alleles indicates a potential physical interaction between the two kinases, which is further supported by coimmunoprecipitation analyses, partial colocalization of both proteins in wild-type cells, and their common mislocalization in dynein/kinesin mutants. We conclude that POD6 acts together with COT1 and is essential for polar cell extension in a kinesin/dynein-dependent manner in N. crassa.

Spastin Interacts with CRMP2 to Regulate Neurite Outgrowth by Controlling Microtubule Dynamics through Phosphorylation Modifications

2020 ◽  
Vol 19 ◽  
Author(s):  
Sumei Li ◽  
Jifeng Zhang ◽  
Jiaqi Zhang ◽  
Jiong Li ◽  
Longfei Cheng ◽  
...  
Keyword(s):  
Neurite Outgrowth ◽  
Hippocampal Neurons ◽  
Neuronal Development ◽  
Phosphorylation Site ◽  
Microtubule Dynamics ◽  
Microtubule Assembly ◽  
C Terminus ◽  
Mediator Protein ◽  
Branch Formation

Aims: Our work aims to revealing the underlying microtubule mechanism of neurites outgrowth during neuronal development, and also proposes a feasible intervention pathway for reconstructing neural network connections after nerve injury. Background: Microtubule polymerization and severing are the basis for the neurite outgrowth and branch formation. Collapsin response mediator protein 2 (CRMP2) regulates axonal growth and branching as a binding partner of the tubulin heterodimer to promote microtubule assembly. And spastin participates in the growth and regeneration of neurites by severing microtubules into small segments. However, how CRMP2 and spastin cooperate to regulate neurite outgrowth by controlling the microtubule dynamics needs to be elucidated. Objective: To explore whether neurite outgrowth was mediated by coordination of CRMP2 and spastin. Method: Hippocampal neurons were cultured in vitro in 24-well culture plates for 4 days before being used to perform the transfection. Calcium phosphate was used to transfect the CRMP2 and spastin constructs and their control into the neurons. An interaction between CRMP2 and spastin was examined by using pull down, CoIP and immunofluorescence colocalization assays. And immunostaining was also performed to determine the morphology of neurites. Result: We first demonstrated that CRMP2 interacted with spastin to promote the neurite outgrowth and branch formation. Furthermore, our results identified that phosphorylation modification failed to alter the binding affinities of CRMP2 for spastin, but inhibited their binding to microtubules. CRMP2 interacted with the MTBD domain of spastin via its C-terminus, and blocking the binding sites of them inhibited the outgrowth and branch formation of neurites. In addition, we confirmed one phosphorylation site S210 at spastin in hippocampal neurons and phosphorylation spastin at site S210 promoted the neurite outgrowth but not branch formation by remodeling microtubules. Conclusion: Taken together, our data demonstrated that the interaction of CRMP2 and spastin is required for neurite outgrowth and branch formation and their interaction is not regulated by their phosphorylation.

scholarly journals Protein Analysis of Pollen Tubes after the Treatments of Membrane Trafficking Inhibitors Gains Insights on Molecular Mechanism Underlying Pollen Tube Polar Growth

2021 ◽  
Vol 40 (2) ◽  
pp. 205-222
Author(s):  
Monica Scali ◽  
Alessandra Moscatelli ◽  
Luca Bini ◽  
Elisabetta Onelli ◽  
Rita Vignani ◽  
...  
Keyword(s):  
Pollen Tube ◽  
Actin Cytoskeleton ◽  
Membrane Trafficking ◽  
Tip Growth ◽  
Male Gametophyte ◽  
Pollen Tubes ◽  
Structural Features ◽  
Two Dimensional Gel Electrophoresis ◽  
Depth Analysis

AbstractPollen tube elongation is characterized by a highly-polarized tip growth process dependent on an efficient vesicular transport system and largely mobilized by actin cytoskeleton. Pollen tubes are an ideal model system to study exocytosis, endocytosis, membrane recycling, and signaling network coordinating cellular processes, structural organization and vesicular trafficking activities required for tip growth. Proteomic analysis was applied to identifyNicotiana tabacumDifferentially Abundant Proteins (DAPs) after in vitro pollen tube treatment with membrane trafficking inhibitors Brefeldin A, Ikarugamycin and Wortmannin. Among roughly 360 proteins separated in two-dimensional gel electrophoresis, a total of 40 spots visibly changing between treated and control samples were identified by MALDI-TOF MS and LC–ESI–MS/MS analysis. The identified proteins were classified according to biological processes, and most proteins were related to pollen tube energy metabolism, including ammino acid synthesis and lipid metabolism, structural features of pollen tube growth as well modification and actin cytoskeleton organization, stress response, and protein degradation. In-depth analysis of proteins corresponding to energy-related pathways revealed the male gametophyte to be a reliable model of energy reservoir and dynamics.

scholarly journals Analysis of Morphokinetic Parameters of Feline Embryos Using a Time-Lapse System

Animals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 748
Author(s):  
Joanna Kochan ◽  
Agnieszka Nowak ◽  
Barbara Kij ◽  
Sylwia Prochowska ◽  
Wojciech Niżański
Keyword(s):  
Embryo Development ◽  
Embryo Quality ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Cavity Formation ◽  
Non Invasive ◽  
Morphokinetic Parameters ◽  
Morphological Defects ◽  
Development In Vitro

The aim of this study was to analyze the morphokinetic parameters of feline embryos using a time lapse system. Oocytes matured in vitro were fertilized (IVF) and in vitro cultured in a time lapse-system (Primo Vision®, Gothenburg, Sweden). The first cell division of embryos occurred between 17 h post insemination (hpi) and 38 hpi, with the highest proportion of embryos (46%) cleaving between 21 and 24 hpi. The timing of the first cleavage significantly affected further embryo development, with the highest development occurring in embryos that cleaved at 21–22 hpi. Embryos that cleaved very early (17–18 hpi) developed poorly to the blastocyst stage (2%) and none of the embryos that cleaved later than 27 hpi were able to reach the blastocyst stage. Morphological defects were observed in 48% of the embryos. There were no statistically significant differences between the timing intervals of the first cleavage division and the frequency of morphological defects in embryos. Multiple (MUL) morphological defects were detected in more than half (56%) of the abnormal embryos. The most frequent single morphological defects were cytoplasmic fragmentation (FR) (8%) and blastomere asymmetry (AS) (6%). Direct cleavage (DC) from 1–3 or 3–5 blastomeres, reverse cleavage (RC) and vacuoles were rarely observed (2–3%). The timing of blastocyst cavity formation is a very good indicator of embryo quality. In our study, blastocyst cavity formation occurred between 127–167 hpi, with the highest frequency of hatching observed in blastocysts that cavitated between 142–150 hpi. Blastocysts in which cavitation began after 161 h did not hatch. In conclusion, the timing of the first and second cleavage divisions, the timing of blastocyst cavity formation and morphological anomalies can all be used as early and non-invasive indicators of cat embryo development in vitro.

scholarly journals Interactions between highly conserved U2 small nuclear RNA structures and Prp5p, Prp9p, Prp11p, and Prp21p proteins are required to ensure integrity of the U2 small nuclear ribonucleoprotein in Saccharomyces cerevisiae.

1994 ◽  
Vol 14 (9) ◽  
pp. 6337-6349 ◽  
Author(s):  
S E Wells ◽  
M Ares
Keyword(s):  
Synthetic Lethality ◽  
Small Nuclear Rna ◽  
Rna Structures ◽  
Stem Loop ◽  
U2 Snrna ◽  
Small Nuclear Ribonucleoprotein ◽  
U2 Snrnp ◽  
Nuclear Rna ◽  
Nuclear Ribonucleoprotein

Binding of U2 small nuclear ribonucleoprotein (snRNP) to the pre-mRNA is an early and important step in spliceosome assembly. We searched for evidence of cooperative function between yeast U2 small nuclear RNA (snRNA) and several genetically identified splicing (Prp) proteins required for the first chemical step of splicing, using the phenotype of synthetic lethality. We constructed yeast strains with pairwise combinations of 28 different U2 alleles with 10 prp mutations and found lethal double-mutant combinations with prp5, -9, -11, and -21 but not with prp3, -4, -8, or -19. Many U2 mutations in highly conserved or invariant RNA structures show no phenotype in a wild-type PRP background but render mutant prp strains inviable, suggesting that the conserved but dispensable U2 elements are essential for efficient cooperative function with specific Prp proteins. Mutant U2 snRNA fails to accumulate in synthetic lethal strains, demonstrating that interaction between U2 RNA and these four Prp proteins contributes to U2 snRNP assembly or stability. Three of the proteins (Prp9p, Prp11p, and Prp21p) are associated with each other and pre-mRNA in U2-dependent splicing complexes in vitro and bind specifically to synthetic U2 snRNA added to crude splicing extracts depleted of endogenous U2 snRNPs. Taken together, the results suggest that Prp9p, -11p, and -21p are U2 snRNP proteins that interact with a structured region including U2 stem loop IIa and mediate the association of the U2 snRNP with pre-mRNA.

Pronounced cytoplasmic pH gradients are not required for tip growth in plant and fungal cells

1997 ◽  
Vol 110 (10) ◽  
pp. 1187-1198 ◽  
Author(s):  
R.M. Parton ◽  
S. Fischer ◽  
R. Malho ◽  
O. Papasouliotis ◽  
T.C. Jelitto ◽  
...  
Keyword(s):  
Tip Growth ◽  
Cell Types ◽  
Pollen Tubes ◽  
Fine Tuning ◽  
Cytoplasmic Ph ◽  
Dual Emission ◽  
Ph Gradients ◽  
Longitudinal Gradients ◽  
External Ph

The existence of pronounced cytoplasmic pH gradients within the apices of tip-growing cells, and the role of cytoplasmic pH in regulating tip growth, were investigated in three different cell types: vegetative hyphae of Neurospora crassa; pollen tubes of Agapanthus umbellatus; and rhizoids of Dryopteris affinis gametophytes. Examination of cytoplasmic pH in growing cells was performed by simultaneous, dual emission confocal ratio imaging of the pH-sensitive probe carboxy SNARF-1. Considerable attention was paid to the fine tuning of dye loading and imaging parameters to minimise cellular perturbation and assess the extent of dye partitioning into organelles. With optimal conditions, cytoplasmic pH was measured routinely with a precision of between +/−0.03 and +/−0.06 of a pH unit and a spatial resolution of 2.3 microm2. Based on in vitro calibration, estimated values of mean cytoplasmic pH for cells loaded with dye-ester were between 7.15 and 7.25 for the three cell types. After pressure injecting Neurospora hyphae with dextran-conjugated dye, however, the mean cytoplasmic pH was estimated to be 7.57. Dextran dyes are believed to give a better estimate of cytoplasmic pH because of their superior localisation and retention within the cytosol. No significant cytoplasmic pH gradient (delta pH of >0.1 unit) was observed within the apical 50 microm in growing cells of any of the three cell types. Acidification or alkalinisation of the cytoplasm in Neurospora hyphae, using a cell permeant weak acid (propionic acid at pH 7.0) or weak base (trimethylamine at pH 8.0), slowed down but did not abolish growth. However, similar manipulation of the cytoplasmic pH of Agapanthus pollen tubes and Dryopteris rhizoids completely inhibited growth. Modification of external pH affected the growth pattern of all cell types. In hyphae and pollen tubes, changes in external pH were found to have a small transient effect on cytoplasmic pH but the cells rapidly readjusted towards their original pH. Our results suggest that pronounced longitudinal gradients in cytoplasmic pH are not essential for the regulation of tip growth.

Morphology of Cell Injury: An Approach to the EDIT Programme by the Use of Tobacco Pollen Tubes

2002 ◽  
Vol 30 (3) ◽  
pp. 323-329 ◽  
Author(s):  
Udo Kristen ◽  
Natalie Bischoff ◽  
Saskia Lisboa ◽  
Enno Schirmer ◽  
Sören Witt ◽  
...  
Keyword(s):  
Tip Growth ◽  
Cell Injury ◽  
Cytosolic Calcium ◽  
Pollen Tubes ◽  
In Vitro Cytotoxicity ◽  
Toxic Compounds ◽  
In Vitro System ◽  
Tobacco Pollen ◽  
Different Types

Tobacco pollen tubes were used as a standard in vitro system to investigate cell growth aberrations caused by some of the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme chemicals and other toxic compounds. Changes in cytoskeletal pattern were observed in the tube cells by using tubu-lin immunofluorescence and rhodamin–phalloidin fluorescence for the localisation of microtubules and actin filaments, respectively. Four different types of cell malformation were found: screw-like growth, isodiametric tip swelling, hook formation, and pollen grain enlargement. We suggest that these malformations resulted from an interference by the chemicals with the cytosolic calcium gradient which controls tip growth and the orientation of the pollen tube. The results may contribute to a general understanding of toxicity-based cell malformations.

scholarly journals Sgs1 Regulates Gene Conversion Tract Lengths and Crossovers Independently of Its Helicase Activity

2006 ◽  
Vol 26 (11) ◽  
pp. 4086-4094 ◽  
Author(s):  
Yi-Chen Lo ◽  
Kimberly S. Paffett ◽  
Or Amit ◽  
Jennifer A. Clikeman ◽  
Rosa Sterk ◽  
...  
Keyword(s):  
Gene Conversion ◽  
Genome Stability ◽  
Synthetic Lethality ◽  
Chromosome Loss ◽  
Helicase Activity ◽  
Tract Length ◽  
Independent Functions ◽  
Gene Conversion Tract ◽  
Conversion Tract

ABSTRACT RecQ helicases maintain genome stability and suppress tumors in higher eukaryotes through roles in replication and DNA repair. The yeast RecQ homolog Sgs1 interacts with Top3 topoisomerase and Rmi1. In vitro, Sgs1 binds to and branch migrates Holliday junctions (HJs) and the human RecQ homolog BLM, with Top3α, resolves synthetic double HJs in a noncrossover sense. Sgs1 suppresses crossovers during the homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Crossovers are associated with long gene conversion tracts, suggesting a model in which Sgs1 helicase catalyzes reverse branch migration and convergence of double HJs for noncrossover resolution by Top3. Consistent with this model, we show that allelic crossovers and gene conversion tract lengths are increased in sgs1Δ. However, crossover and tract length suppression was independent of Sgs1 helicase activity, which argues against helicase-dependent HJ convergence. HJs may converge passively by a “random walk,” and Sgs1 may play a structural role in stimulating Top3-dependent resolution. In addition to the new helicase-independent functions for Sgs1 in crossover and tract length control, we define three new helicase-dependent functions, including the suppression of chromosome loss, chromosome missegregation, and synthetic lethality in srs2Δ. We propose that Sgs1 has helicase-dependent functions in replication and helicase-independent functions in DSB repair by HR.

scholarly journals Individual Mycobacterium tuberculosis Resuscitation-Promoting Factor Homologues Are Dispensable for Growth In Vitro and In Vivo

2004 ◽  
Vol 72 (1) ◽  
pp. 515-526 ◽  
Author(s):  
JoAnn M. Tufariello ◽  
William R. Jacobs, ◽  
John Chan
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Stationary Phase ◽  
Essential Gene ◽  
Micrococcus Luteus ◽  
Expression Patterns ◽  
Active Growth ◽  
Prolonged Incubation ◽  
Aerosol Infection

ABSTRACT Mycobacterium tuberculosis possesses five genes with significant homology to the resuscitation-promoting factor (Rpf) of Micrococcus luteus. The M. luteus Rpf is a secreted ∼16-kDa protein which restores active growth to cultures of M. luteus rendered dormant by prolonged incubation in stationary phase. More recently, the Rpf-like proteins of M. tuberculosis have been shown to stimulate the growth of extended-stationary-phase cultures of Mycobacterium bovis BCG. These data suggest that the Rpf proteins can influence the growth of mycobacteria; however, the studies do not demonstrate specific functions for the various members of this protein family, nor do they assess the function of M. tuberculosis Rpf homologues in vivo. To address these questions, we have disrupted each of the five rpf-like genes in M. tuberculosis Erdman, and analyzed the mutants for their growth in vitro and in vivo. In contrast to M. luteus, for which rpf is an essential gene, we find that all of the M. tuberculosis rpf deletion mutant strains are viable; in addition, all show growth kinetics similar to Erdman wild type both in vitro and in mouse organs following aerosol infection. Analysis of rpf expression in M. tuberculosis cultures from early log phase through late stationary phase indicates that expression of the rpf-like genes is growth phase-dependent, and that the expression patterns of the five M. tuberculosis rpf genes, while overlapping to various degrees, are not uniform. We also provide evidence that mycobacterial rpf genes are expressed in vivo in the lungs of mice acutely infected with virulent M. tuberculosis.

scholarly journals The Formin mDia Regulates GSK3β through Novel PKCs to Promote Microtubule Stabilization but Not MTOC Reorientation in Migrating Fibroblasts

2006 ◽  
Vol 17 (12) ◽  
pp. 5004-5016 ◽  
Author(s):  
Christina H. Eng ◽  
Thomas M. Huckaba ◽  
Gregg G. Gundersen
Keyword(s):  
Rho Gtpase ◽  
Glycogen Synthase ◽  
Dominant Negative ◽  
Microtubule Stabilization ◽  
3T3 Fibroblasts ◽  
Organizing Center ◽  
Inhibitory Site ◽  
External Signals

In migrating cells, external signals polarize the microtubule (MT) cytoskeleton by stimulating the formation of oriented, stabilized MTs and inducing the reorientation of the MT organizing center (MTOC). Glycogen synthase kinase 3β (GSK3β) has been implicated in each of these processes, although whether it regulates both processes in a single system and how its activity is regulated are unclear. We examined these issues in wound-edge, serum-starved NIH 3T3 fibroblasts where MT stabilization and MTOC reorientation are triggered by lysophosphatidic acid (LPA), but are regulated independently by distinct Rho GTPase-signaling pathways. In the absence of other treatments, the GSK3β inhibitors, LiCl or SB216763, induced the formation of stable MTs, but not MTOC reorientation, in starved fibroblasts. Overexpression of GSK3β in starved fibroblasts inhibited LPA-induced stable MTs without inhibiting MTOC reorientation. Analysis of factors involved in stable MT formation (Rho, mDia, and EB1) showed that GSK3β functioned upstream of EB1, but downstream of Rho-mDia. mDia was both necessary and sufficient for inducing stable MTs and for up-regulating GSK3β phosphorylation on Ser9, an inhibitory site. mDia appears to regulate GSK3β through novel class PKCs because PKC inhibitors and dominant negative constructs of novel PKC isoforms prevented phosphorylation of GSK3β Ser9 and stable MT formation. Novel PKCs also interacted with mDia in vivo and in vitro. These results identify a new activity for the formin mDia in regulating GSK3β through novel PKCs and implicate novel PKCs as new factors in the MT stabilization pathway.

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