Myosin XI drives polarized growth by vesicle focusing and local enrichment of F-actin in Physcomitrium patens

2021 ◽  
Author(s):  
Giulia Galotto ◽  
Pattipong Wisanpitayakorn ◽  
Jeffrey P Bibeau ◽  
Yen-Chun Liu ◽  
Fabienne Furt ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Function Analysis ◽  
Temperature Sensitive ◽  
Local Concentration ◽  
Loss Of Function ◽  
Cell Wall Polysaccharides ◽  
Actin Organization ◽  
Myosin Xi ◽  
Sensitive Allele ◽  
Local Enrichment

Abstract In tip-growing plant cells, growth results from myosin XI and F-actin-mediated deposition of cell wall polysaccharides contained in secretory vesicles. Previous evidence showed that myosin XI anticipates F-actin accumulation at the cell’s tip, suggesting a mechanism where vesicle clustering via myosin XI increases F-actin polymerization. To evaluate this model, we used a conditional loss-of-function strategy by generating moss (Physcomitrium patens) plants harboring a myosin XI temperature-sensitive allele. We found that loss of myosin XI function alters tip cell morphology, vacuolar homeostasis, and cell viability but not following F-actin depolymerization. Importantly, our conditional loss-of-function analysis shows that myosin XI focuses and directs vesicles at the tip of the cell, which induces formin-dependent F-actin polymerization, increasing F-actin’s local concentration. Our findings support the role of myosin XI in vesicle focusing, possibly via clustering and F-actin organization, necessary for tip growth, and deepen our understanding of additional myosin XI functions.

scholarly journals “Myosin XI drives polarized growth by vesicle clustering and local enrichment of F-actin in Physcomitrium (Physcomitrella) patens”

2020 ◽  
Author(s):  
Giulia Galotto ◽  
Pattipong Wisanpitayakorn ◽  
Jeffrey P. Bibeau ◽  
Yen-Chun Liu ◽  
Parker J. Simpson ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Physcomitrella Patens ◽  
Function Analysis ◽  
Temperature Sensitive ◽  
Local Concentration ◽  
Loss Of Function ◽  
Cell Wall Polysaccharides ◽  
Actin Organization ◽  
Myosin Xi ◽  
Local Enrichment

ABSTRACTIn tip-growing plant cells, growth results from myosin XI and F-actin mediated deposition of cell wall polysaccharides contained in secretory vesicles. Previous evidence showed that myosin XI anticipates F-actin accumulation at the cell’s tip, suggesting a mechanism where vesicle clustering via myosin XI promotes F-actin polymerization. To evaluate this model, we used a conditional loss-of-function strategy by generating Physcomitrium (Physcomitrella) patens plants harboring a myosin XI temperature-sensitive allele. We found that loss of myosin XI function alters tip cell morphology, vacuolar homeostasis, and cell viability, but not following F-actin depolymerization. Importantly, our conditional loss-of-function analysis shows that myosin XI clusters and directs vesicles at the tip of the cell, which induces F-actin polymerization, increasing F-actin’s local concentration. Our findings support the role of myosin XI in vesicle clustering and F-actin organization, necessary for tip growth, and deepen our understanding of additional myosin XI functions.

scholarly journals Negative Regulation of Yeast Eps15-like Arp2/3 Complex Activator, Pan1p, by the Hip1R-related Protein, Sla2p, during Endocytosis

2007 ◽  
Vol 18 (2) ◽  
pp. 658-668 ◽  
Author(s):  
Jiro Toshima ◽  
Junko Y. Toshima ◽  
Mara C. Duncan ◽  
M. Jamie T.V. Cope ◽  
Yidi Sun ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Affinity Purification ◽  
Coiled Coil ◽  
Actin Assembly ◽  
Related Protein ◽  
Loss Of Function ◽  
Actin Organization ◽  
Coiled Coil Region ◽  
Kinase Phosphorylation

Control of actin assembly nucleated by the Arp2/3 complex plays a crucial role during budding yeast endocytosis. The yeast Eps15-related Arp2/3 complex activator, Pan1p, is essential for endocytic internalization and proper actin organization. Pan1p activity is negatively regulated by Prk1 kinase phosphorylation after endocytic internalization. Phosphorylated Pan1p is probably then dephosphorylated in the cytosol. Pan1p is recruited to endocytic sites ∼25 s before initiation of actin polymerization, suggesting that its Arp2/3 complex activation activity is kept inactive during early stages of endocytosis by a yet-to-be-identified mechanism. However, how Pan1p is maintained in an inactive state is not clear. Using tandem affinity purification–tagged Pan1p, we identified End3p as a stoichiometric component of the Pan1p complex, and Sla2p, a yeast Hip1R-related protein, as a novel binding partner of Pan1p. Interestingly, Sla2p specifically inhibited Pan1p Arp2/3 complex activation activity in vitro. The coiled-coil region of Sla2p was important for Pan1p inhibition, and a pan1 partial loss-of-function mutant suppressed the temperature sensitivity, endocytic phenotypes, and actin phenotypes observed in sla2ΔCC mutant cells that lack the coiled-coil region. Overall, our results establish that Sla2p's regulation of Pan1p plays an important role in controlling Pan1p-stimulated actin polymerization during endocytosis.

scholarly journals Role of Cdc42p in Pheromone-Stimulated Signal Transduction in Saccharomyces cerevisiae

2000 ◽  
Vol 20 (20) ◽  
pp. 7559-7571 ◽  
Author(s):  
John J. Moskow ◽  
Amy S. Gladfelter ◽  
Rachel E. Lamson ◽  
Peter M. Pryciak ◽  
Daniel J. Lew
Keyword(s):  
Signal Transduction ◽  
Mammalian Cells ◽  
Mitogen Activated Protein Kinase ◽  
Temperature Sensitive ◽  
Local Concentration ◽  
Pheromone Response ◽  
Actin Organization ◽  
Epistasis Analysis

ABSTRACT CDC42 encodes a highly conserved GTPase of the Rho family that is best known for its role in regulating cell polarity and actin organization. In addition, various studies of both yeast and mammalian cells have suggested that Cdc42p, through its interaction with p21-activated kinases (PAKs), plays a role in signaling pathways that regulate target gene transcription. However, recent studies of the yeast pheromone response pathway suggested that prior results with temperature-sensitive cdc42 mutants were misleading and that Cdc42p and the Cdc42p-PAK interaction are not involved in signaling. To clarify this issue, we have identified and characterized novel viable pheromone-resistant cdc42 alleles that retain the ability to perform polarity-related functions. Mutation of the Cdc42p residue Val36 or Tyr40 caused defects in pheromone signaling and in the localization of the Ste20p PAK in vivo and affected binding to the Ste20p Cdc42p-Rac interactive binding (CRIB) domain in vitro. Epistasis analysis suggested that they affect the signaling step at which Ste20p acts, and overproduction of Ste20p rescued the defect. These results suggest that Cdc42p is in fact required for pheromone response and that interaction with the PAK Ste20p is critical for that role. Furthermore, the ste20ΔCRIB allele, previously used to disrupt the Cdc42p-Ste20p interaction, behaved as an activated allele, largely bypassing the signaling defect of thecdc42 mutants. Additional observations lead us to suggest that Cdc42p collaborates with the SH3-domain protein Bem1p to facilitate signal transduction, possibly by providing a cell surface scaffold that aids in the local concentration of signaling kinases, thus promoting activation of a mitogen-activated protein kinase cascade by Ste20p.

gon-2, a Gene Required for Gonadogenesis in Caenorhabditis elegans

Genetics ◽  
1997 ◽  
Vol 147 (3) ◽  
pp. 1077-1089 ◽  
Author(s):  
Andrew Y Sun ◽  
Eric J Lambie
Keyword(s):  
Caenorhabditis Elegans ◽  
Larval Stage ◽  
Cell Lineage ◽  
Temperature Sensitive ◽  
Loss Of Function ◽  
Primary Defect ◽  
Cell Fates ◽  
Fourth Larval Stage ◽  
Full Complement ◽  
Sensitive Allele

The gonad of the Caenorhabditis elegans hermaphrodite is generated by the postembryonic divisions of two somatic precursors, Z1 and Z4, and two germline precursors, Z2 and Z3. These cells begin division midway through the first larval stage. By the end of the fourth larval stage, Z1 and Z4 produce 143 descendants, while Z2 and Z3 give rise to ∼1000 descendants. The divisions of Z2 and Z3 are dependent on signals produced by Z1 and Z4, but not vice versa. We have characterized the properties of five loss-of-function alleles of a newly described gene, which we call gon-2. In gon-2 mutants, gonadogenesis is severely impaired; in some animals, none of the gonad progenitors undergo any postembryonic divisions. Mutations in gon-2 have a partial maternal effect: either maternal or zygotic expression is sufficient to prevent the severe gonadogenesis defects. By cell lineage analysis, we found that the primary defect in gon-2 mutants is a delay (sometimes a complete block) in the onset and continuation of gonadal divisions. The results of upshift experiments using a temperature-sensitive allele suggest that zygotic expression of gon-2 begins early in embryogenesis, before the birth of Z1 and Z4. The results of downshift experiments suggest that Z1 and Z4 can generate the full complement of gonadal tissues even when gon-2 function is inhibited until the end of the second larval stage. Thus, gon-2 activity is probably not required for the specification of gonadal cell fates, but appears to be generally required for gonadal cell divisions.

scholarly journals Identification and Characterization of Genes That Interact With lin-12 in Caenorhabditis elegans

Genetics ◽  
1997 ◽  
Vol 147 (4) ◽  
pp. 1675-1695 ◽  
Author(s):  
Frans E Tax ◽  
James H Thomas ◽  
Edwin L Ferguson ◽  
H Robert Horvitzt
Keyword(s):  
Cell Fate ◽  
Low Frequency ◽  
Signal Transduction Pathway ◽  
Temperature Shift ◽  
Egg Laying ◽  
Null Alleles ◽  
Temperature Sensitive ◽  
Loss Of Function ◽  
Gain Of Function ◽  
Suppressor Mutations

Abstract We identified and characterized 14 extragenic mutations that suppressed the dominant egg-laying defect of certain lin-12 gain-of-function mutations. These suppressors defined seven genes: sup-l7, lag-2, sel-4, sel-5, sel-6, sel-7 and sel-8. Mutations in six of the genes are recessive suppressors, whereas the two mutations that define the seventh gene, lag-2, are semi-dominant suppressors. These suppressor mutations were able to suppress other lin-12 gain-of-function mutations. The suppressor mutations arose at a very low frequency per gene, 10-50 times below the typical loss-of-function mutation frequency. The suppressor mutations in sup1 7 and lag-2 were shown to be rare non-null alleles, and we present evidence that null mutations in these two genes cause lethality. Temperature-shift studies for two suppressor genes, sup1 7and lag-2, suggest that both genes act at approximately the same time as lin-12in specifying a cell fate. Suppressor alleles of six of these genes enhanced a temperature-sensitive loss-of-function allele of glp-1, a gene related to lin-12 in structure and function. Our analysis of these suppressors suggests that the majority of these genes are part of a shared lin-12/glp-1 signal transduction pathway, or act to regulate the expression or stability of lin-12 and glp-1.

scholarly journals Tropomyosin inhibits ADF/cofilin-dependent actin filament dynamics

2002 ◽  
Vol 156 (6) ◽  
pp. 1065-1076 ◽  
Author(s):  
Shoichiro Ono ◽  
Kanako Ono
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Biological Properties ◽  
Actin Dynamics ◽  
Background Suppression ◽  
Thin Filaments ◽  
Actin Organization ◽  
C Elegans ◽  
Filament Dynamics

Tropomyosin binds to actin filaments and is implicated in stabilization of actin cytoskeleton. We examined biochemical and cell biological properties of Caenorhabditis elegans tropomyosin (CeTM) and obtained evidence that CeTM is antagonistic to ADF/cofilin-dependent actin filament dynamics. We purified CeTM, actin, and UNC-60B (a muscle-specific ADF/cofilin isoform), all of which are derived from C. elegans, and showed that CeTM and UNC-60B bound to F-actin in a mutually exclusive manner. CeTM inhibited UNC-60B–induced actin depolymerization and enhancement of actin polymerization. Within isolated native thin filaments, actin and CeTM were detected as major components, whereas UNC-60B was present at a trace amount. Purified UNC-60B was unable to interact with the native thin filaments unless CeTM and other associated proteins were removed by high-salt extraction. Purified CeTM was sufficient to restore the resistance of the salt-extracted filaments from UNC-60B. In muscle cells, CeTM and UNC-60B were localized in different patterns. Suppression of CeTM by RNA interference resulted in disorganized actin filaments and paralyzed worms in wild-type background. However, in an ADF/cofilin mutant background, suppression of CeTM did not worsen actin organization and worm motility. These results suggest that tropomyosin is a physiological inhibitor of ADF/cofilin-dependent actin dynamics.

scholarly journals Profilin-1; a novel regulator of DNA damage response and repair machinery in keratinocytes

2021 ◽  
Author(s):  
Chang-Jin Lee ◽  
Min-Ji Yoon ◽  
Dong Hyun Kim ◽  
Tae Uk Kim ◽  
Youn-Jung Kang
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Recovery Time ◽  
Actin Polymerization ◽  
Loss Of Function ◽  
Dna Double Strand Breaks ◽  
Pten Loss ◽  
Damage Response ◽  
Specific Regulation

AbstractProfilin-1 (PFN1) regulates actin polymerization and cytoskeletal growth. Despite the essential roles of PFN1 in cell integration, its subcellular function in keratinocyte has not been elucidated yet. Here we characterize the specific regulation of PFN1 in DNA damage response and repair machinery. PFN1 depletion accelerated DNA damage-mediated apoptosis exhibiting PTEN loss of function instigated by increased phosphorylated inactivation followed by high levels of AKT activation. PFN1 changed its predominant cytoplasmic localization to the nucleus upon DNA damage and subsequently restored the cytoplasmic compartment during the recovery time. Even though γH2AX was recruited at the sites of DNA double strand breaks in response to DNA damage, PFN1-deficient cells failed to recruit DNA repair factors, whereas control cells exhibited significant increases of these genes. Additionally, PFN1 depletion resulted in disruption of PTEN-AKT cascade upon DNA damage and CHK1-mediated cell cycle arrest was not recovered even after the recovery time exhibiting γH2AX accumulation. This might suggest PFN1 roles in regulating DNA damage response and repair machinery to protect cells from DNA damage. Future studies addressing the crosstalk and regulation of PTEN-related DNA damage sensing and repair pathway choice by PFN1 may further aid to identify new mechanistic insights for various DNA repair disorders.

The Mitochondrial Nucleoid Protein, Mgm101p, of Saccharomyces cerevisiae Is Involved in the Maintenance of ρ+ and ori/rep-Devoid Petite Genomes but Is Not Required for Hypersuppressive ρ− mtDNA

Genetics ◽  
2002 ◽  
Vol 160 (4) ◽  
pp. 1389-1400
Author(s):  
Xiao Ming Zuo ◽  
G Desmond Clark-Walker ◽  
Xin Jie Chen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Mitochondrial Rna ◽  
Mtdna Copy Number ◽  
Mitochondrial Rna Polymerase ◽  
Mitochondrial Nucleoids ◽  
Mtdna Replication ◽  
Sensitive Allele ◽  
Discriminatory Effect

Abstract The Saccharomyces cerevisiae MGM101 gene encodes a DNA-binding protein targeted to mitochondrial nucleoids. MGM101 is essential for maintenance of a functional ρ+ genome because meiotic segregants, with a disrupted mgm101 allele, cannot undergo more than 10 divisions on glycerol medium. Quantitative analysis of mtDNA copy number in a ρ+ strain carrying a temperature-sensitive allele, mgm101-1, revealed that the amount of mtDNA is halved each cell division upon a shift to the restrictive temperature. These data suggest that mtDNA replication is rapidly blocked in cells lacking MGM101. However, a small proportion of meiotic segregants, disrupted in MGM101, have ρ− genomes that are stably maintained. Interestingly, all surviving ρ− mtDNAs contain an ori/rep sequence. Disruption of MGM101 in hypersuppressive (HS) strains does not have a significant effect on the propagation of HS ρ− mtDNA. However, in petites lacking an ori/rep, disruption of MGM101 leads to either a complete loss or a dramatically decreased stability of mtDNA. This discriminatory effect of MGM101 suggests that replication of ρ+ and ori/rep-devoid ρ− mtDNAs is carried out by the same process. By contrast, the persistence of ori/rep-containing mtDNA in HS petites lacking MGM101 identifies a distinct replication pathway. The alternative mtDNA replication mechanism provided by ori/rep is independent of mitochondrial RNA polymerase encoded by RPO41 as a HS ρ− genome is stably maintained in a mgm101, rpo41 double mutant.

Development of the Drosophila olfactory sense organs utilizes cell-cell interactions as well as lineage

Development ◽  
1997 ◽  
Vol 124 (3) ◽  
pp. 703-712 ◽  
Author(s):  
G.V. Reddy ◽  
B. Gupta ◽  
K. Ray ◽  
V. Rodrigues
Keyword(s):  
Cell Communication ◽  
Cell Number ◽  
Temperature Sensitive ◽  
Sense Organs ◽  
Notch Activity ◽  
Helix Loop Helix ◽  
Olfactory Sense ◽  
Sensitive Allele ◽  
Selection Of ◽  
Cell Cell

We have examined the mechanisms underlying the development of the olfactory sense organs on the third segment of the antenna of Drosophila. Our studies suggest that a novel developmental strategy is employed. Specification of the founder or precursor cell is not governed by the genes of the achaete-scute complex. Another basic helix-loop-helix encoding gene, atonal, is essential for determination of only a subset of the sensilla types--the sensilla coeloconica. Therefore, we predict the existence of additional proneural genes for the selection of sensilla trichoidea and sensilla basiconica. The choice of a founder cell from the presumed proneural domain is regulated by Notch activity. Soon after delamination of the founder cell, two to three additional neighboring cells also take on a sensory fate and these cells together form a presensillum cluster. The selection of neighbors does not occur when endocytosis is blocked using a temperature sensitive allele of shibire, thus suggesting that cell-cell communication is required for this step. The cells of the cluster divide once before terminal differentiation which is influenced by Notch activity. The final cell number within each sensillum is controlled by programmed cell death.

scholarly journals Casein kinase I-like protein kinases encoded by YCK1 and YCK2 are required for yeast morphogenesis.

1993 ◽  
Vol 13 (5) ◽  
pp. 2870-2881 ◽  
Author(s):  
L C Robinson ◽  
M M Menold ◽  
S Garrett ◽  
M R Culbertson
Keyword(s):  
Protein Kinase ◽  
Eukaryotic Cell ◽  
Casein Kinase ◽  
Temperature Sensitive ◽  
Protein Kinase Activity ◽  
Restrictive Temperature ◽  
Loss Of Function ◽  
Casein Kinase I ◽  
Yeast Saccharomyces Cerevisiae

Casein kinase I is an acidotropic protein kinase class that is widely distributed among eukaryotic cell types. In the yeast Saccharomyces cerevisiae, the casein kinase I isoform encoded by the gene pair YCK1 and YCK2 is a 60- to 62-kDa membrane-associated form. The Yck proteins perform functions essential for growth and division; either alone supports growth, but loss of function of both is lethal. We report here that casein kinase I-like activity is associated with a soluble Yck2-beta-galactosidase fusion protein in vitro and that thermolabile protein kinase activity is exhibited by a protein encoded by fusion of a temperature-sensitive yck2 allele with lacZ. Cells carrying the yck2-2ts allele arrest at restrictive temperature with multiple, elongated buds containing multiple nuclei. This phenotype suggests that the essential functions of the Yck proteins include roles in bud morphogenesis, possibly in control of cell growth polarity, and in cytokinesis or cell separation. Further, a genetic relationship between the yck2ts allele and deletion of CDC55 indicates that the function of Yck phosphorylation may be related to that of protein phosphatase 2A activity.

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