scholarly journals On the Role of Myosin-II in Cytokinesis: Division ofDictyostelium Cells under Adhesive and Nonadhesive Conditions

1997 ◽  
Vol 8 (12) ◽  
pp. 2617-2629 ◽  
Author(s):  
Ji-Hong Zang ◽  
Guy Cavet ◽  
James H. Sabry ◽  
Peter Wagner ◽  
Sheri L. Moores ◽  
...  
Keyword(s):  
Cell Division ◽  
Light Chain ◽  
Binding Sites ◽  
Myosin Ii ◽  
Hydrophobic Surface ◽  
Wild Type ◽  
Daughter Cells ◽  
Adhesive Surface ◽  
Spatial And Temporal Changes

We have investigated the role of myosin in cytokinesis inDictyostelium cells by examining cells under both adhesive and nonadhesive conditions. On an adhesive surface, both wild-type and myosin-null cells undergo the normal processes of mitotic rounding, cell elongation, polar ruffling, furrow ingression, and separation of daughter cells. When cells are denied adhesion through culturing in suspension or on a hydrophobic surface, wild-type cells undergo these same processes. However, cells lacking myosin round up and polar ruffle, but fail to elongate, furrow, or divide. These differences show that cell division can be driven by two mechanisms that we term Cytokinesis A, which requires myosin, and Cytokinesis B, which is cell adhesion dependent. We have used these approaches to examine cells expressing a myosin whose two light chain-binding sites were deleted (ΔBLCBS-myosin). Although this myosin is a slower motor than wild-type myosin and has constitutively high activity due to the abolition of regulation by light-chain phosphorylation, cells expressing ΔBLCBS-myosin were previously shown to divide in suspension ( Uyeda et al., 1996 ). However, we suspected their behavior during cytokinesis to be different from wild-type cells given the large alteration in their myosin. Surprisingly, ΔBLCBS-myosin undergoes relatively normal spatial and temporal changes in localization during mitosis. Furthermore, the rate of furrow progression in cells expressing a ΔBLCBS-myosin is similar to that in wild-type cells.

scholarly journals Phosphorylation of myosin II regulatory light chain is necessary for migration of HeLa cells but not for localization of myosin II at the leading edge

2003 ◽  
Vol 370 (2) ◽  
pp. 551-556 ◽  
Author(s):  
Katsumi FUMOTO ◽  
Takashi UCHIMURA ◽  
Takahiro IWASAKI ◽  
Kozue UEDA ◽  
Hiroshi HOSOYA
Keyword(s):  
Light Chain ◽  
Hela Cells ◽  
Myosin Ii ◽  
Leading Edge ◽  
Regulatory Light Chain ◽  
Wild Type ◽  
Wound Healing Assay ◽  
Cell Monolayers ◽  
Indirect Immunofluorescence Staining

To investigate the role of phosphorylated myosin II regulatory light chain (MRLC) in living cell migration, these mutant MRLCs were engineered and introduced into HeLa cells. The mutant MRLCs include an unphosphorylatable form, in which both Thr-18 and Ser-19 were substituted with Ala (AA-MRLC), and pseudophosphorylated forms, in which Thr-18 and Ser-19 were replaced with Ala and Asp, respectively (AD-MRLC), and both Thr-18 and Ser-19 were replaced with Asp (DD-MRLC). Mutant MRLC-expressing cell monolayers were mechanically stimulated by scratching, and the cells were forced to migrate in a given direction. In this wound-healing assay, the AA-MRLC-expressing cells migrated much more slowly than the wild-type MRLC-expressing cells. In the case of DD-MRLC- and AD-MRLC-expressing cells, no significant differences compared with wild-type MRLC-expressing cells were observed in their migration speed. Indirect immunofluorescence staining showed that the accumulation of endogenous diphosphorylated MRLC at the leading edge was not observed in AA-MRLC-expressing cells, although AA-MRLC was incorporated into myosin heavy chain and localized at the leading edge. In conclusion, we propose that the phosphorylation of MRLC is required to generate the driving force in the migration of the cells but not necessary for localization of myosin II at the leading edge.

scholarly journals Novel Myosin Heavy Chain Kinase Involved in Disassembly of Myosin II Filaments and Efficient Cleavage in MitoticDictyostelium Cells

2002 ◽  
Vol 13 (12) ◽  
pp. 4333-4342 ◽  
Author(s):  
Akira Nagasaki ◽  
Go Itoh ◽  
Shigehiko Yumura ◽  
Taro Q.P. Uyeda
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Myosin Ii ◽  
Kinase Domain ◽  
Cleavage Furrow ◽  
Wild Type ◽  
Daughter Cells ◽  
Cleavage Process ◽  
Interphase Cells ◽  
Myosin Heavy Chain Kinase

We have cloned a full-length cDNA encoding a novel myosin II heavy chain kinase (mhckC) from Dictyostelium. Like other members of the myosin heavy chain kinase family, themhckC gene product, MHCK C, has a kinase domain in its N-terminal half and six WD repeats in the C-terminal half. GFP-MHCK C fusion protein localized to the cortex of interphase cells, to the cleavage furrow of mitotic cells, and to the posterior of migrating cells. These distributions of GFP-MHCK C always corresponded with that of myosin II filaments and were not observed in myosin II-null cells, where GFP-MHCK C was diffusely distributed in the cytoplasm. Thus, localization of MHCK C seems to be myosin II-dependent. Cells lacking the mhckC gene exhibited excessive aggregation of myosin II filaments in the cleavage furrows and in the posteriors of the daughter cells once cleavage was complete. The cleavage process of these cells took longer than that of wild-type cells. Taken together, these findings suggest MHCK C drives the disassembly of myosin II filaments for efficient cytokinesis and recycling of myosin II that occurs during cytokinesis.

Abstract MP20: Optogenetic Control Of Pip2 To Assess Mechanisms Regulating The Epithelial Sodium Channel

Hypertension ◽  
2021 ◽  
Vol 78 (Suppl_1) ◽  
Author(s):  
Crystal R Archer ◽  
Amanpreet Kaur ◽  
Tarek Mohamed ◽  
James D Stockand
Keyword(s):  
Binding Sites ◽  
Hek293 Cells ◽  
Proper Function ◽  
Small Decrease ◽  
Kidney Tubules ◽  
Wild Type ◽  
N Terminus ◽  
Patch Clamp Electrophysiology ◽  
G Protein Coupled

The epithelial Na + channel (ENaC) plays a key role in Na + transport in epithelial linings to include the lung, colon and kidney. In the distal kidney tubules, ENaC regulates Na + reabsorption and blood volume. Thus, dysfunctions in signaling pathways regulating ENaC activity are linked to hypertension or hypotension. Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) is a target of the G protein coupled receptor P2Y2 pathway, and is necessary for the proper function of ENaC. This nonvoltage-gated trimeric channel is comprised of α, β, and γ subunits. We recently described two intracellular PIP 2 binding sites on the N termini of β-, and γ-ENaC, with moderate μM affinity. Here, we report the functional effects on ENaC containing a combination of mutations to those PIP 2 binding sites, by controlled depletion of PIP 2 . We used a CIBN/CRY2-5-ptase optogenetic dimerization system to deplete PIP 2 levels in HEK293 cells transiently expressing wild type (wt) ENaC or the mutant ENaC constructs. A fluorescent Na + indicator, was used to monitor ENaC activity by tracking the relative intracellular Na + levels. Upon optogenetic-controlled depletion of PIP 2 , Na + levels decreased in cells expressing wt ENaC. Mutations to the PIP 2 sites of ENaC were expected to have no change in Na + levels upon PIP 2 depletion due to the disruption of PIP 2 binding. As a control, mutations to non-PIP 2 binding sites were included, and were expected to have decreased Na + levels similar to wt ENaC. Interestingly, mutation of each independent PIP 2 site resulted in only a small decrease of intracellular Na + , compared to wt ENaC. However, mutations throughout the entire N-terminus of β-ENaC, including the PIP 2 binding site, resulted in a significant increase of Na + upon PIP 2 depletion. We performed patch clamp electrophysiology and found that the ENaC recordings corresponded to the Na + fluctuations. These data suggest that the residues surrounding the PIP 2 binding sites play a significant role in the affinity of PIP 2 for ENaC. The role of these other domains in PIP 2 binding is still under investigation.

scholarly journals Deletion of 1–43 amino acids in cardiac myosin essential light chain blunts length dependency of Ca2+ sensitivity and cross-bridge detachment kinetics

2013 ◽  
Vol 304 (2) ◽  
pp. H253-H259 ◽  
Author(s):  
John Jeshurun Michael ◽  
Sampath K. Gollapudi ◽  
Steven J. Ford ◽  
Katarzyna Kazmierczak ◽  
Danuta Szczesna-Cordary ◽  
...  
Keyword(s):  
Amino Acids ◽  
Light Chain ◽  
Sarcomere Length ◽  
Cardiac Myosin ◽  
Wild Type ◽  
Essential Light Chain ◽  
Cross Bridge ◽  
Maximal Activation ◽  
Cardiac Muscle Fibers

The role of cardiac myosin essential light chain (ELC) in the sarcomere length (SL) dependency of myofilament contractility is unknown. Therefore, mechanical and dynamic contractile properties were measured at SL 1.9 and 2.2 μm in cardiac muscle fibers from two groups of transgenic (Tg) mice: 1) Tg-wild-type (WT) mice that expressed WT human ventricular ELC and 2) Tg-Δ43 mice that expressed a mutant ELC lacking 1–43 amino acids. In agreement with previous studies, Ca2+-activated maximal tension decreased significantly in Tg-Δ43 fibers. pCa50 (−log10 [Ca2+]free required for half maximal activation) values at SL of 1.9 μm were 5.64 ± 0.02 and 5.70 ± 0.02 in Tg-WT and Tg-Δ43 fibers, respectively. pCa50 values at SL of 2.2 μm were 5.70 ± 0.01 and 5.71 ± 0.01 in Tg-WT and Tg-Δ43 fibers, respectively. The SL-mediated increase in the pCa50 value was statistically significant only in Tg-WT fibers ( P < 0.01), indicating that the SL dependency of myofilament Ca2+ sensitivity was blunted in Tg-Δ43 fibers. The SL dependency of cross-bridge (XB) detachment kinetics was also blunted in Tg-Δ43 fibers because the decrease in XB detachment kinetics was significant ( P < 0.001) only at SL 1.9 μm. Thus the increased XB dwell time at the short SL augments Ca2+ sensitivity at short SL and thus blunts SL-mediated increase in myofilament Ca2+ sensitivity. Our data suggest that the NH2-terminal extension of cardiac ELC not only augments the amplitude of force generation, but it also may play a role in mediating the SL dependency of XB detachment kinetics and myofilament Ca2+ sensitivity.

scholarly journals Landscape of Resistance-Nodulation-Cell Division (RND)-Type Efflux Pumps in Enterobacter cloacae Complex

2016 ◽  
Vol 60 (4) ◽  
pp. 2373-2382 ◽  
Author(s):  
François Guérin ◽  
Claire Lallement ◽  
Christophe Isnard ◽  
Anne Dhalluin ◽  
Vincent Cattoir ◽  
...  
Keyword(s):  
Cell Division ◽  
Antimicrobial Resistance ◽  
Galleria Mellonella ◽  
Acquired Resistance ◽  
Enterobacter Cloacae ◽  
Efflux Pumps ◽  
Wild Type ◽  
Content Type ◽  
Active Efflux

ABSTRACTIn Gram-negative bacteria, the active efflux is an important mechanism of antimicrobial resistance, but little is known about theEnterobacter cloacaecomplex (ECC). It is mediated primarily by pumps belonging to the RND (resistance-nodulation-cell division) family, and only AcrB, part of the AcrAB-TolC tripartite system, was characterized in ECC. However, detailed genome sequence analysis of the strainE. cloacaesubsp.cloacaeATCC 13047 revealed to us that 10 other genes putatively coded for RND-type transporters. We then characterized the role of all of these candidates by construction of corresponding deletion mutants, which were tested for their antimicrobial susceptibility to 36 compounds, their virulence in the invertebrateGalleria mellonellamodel of infection, and their ability to form biofilm. Only the ΔacrBmutant displayed significantly different phenotypes compared to that of the wild-type strain: 4- to 32-fold decrease of MICs of several antibiotics, antiseptics, and dyes, increased production of biofilm, and attenuated virulence inG. mellonella. In order to identify specific substrates of each pump, we individually expressed intransall operons containing an RND pump-encoding gene into the ΔacrBhypersusceptible strain. We showed that three other RND-type efflux systems (ECL_00053-00055, ECL_01758-01759, and ECL_02124-02125) were able to partially restore the wild-type phenotype and to superadd to and even enlarge the broad range of antimicrobial resistance. This is the first global study assessing the role of all RND efflux pumps chromosomally encoded by the ECC, which confirms the major role of AcrB in both pathogenicity and resistance and the potential involvement of other RND-type members in acquired resistance.

scholarly journals The Escherichia coli datA site promotes proper regulation of cell division

Microbiology ◽  
2014 ◽  
Vol 160 (4) ◽  
pp. 703-710 ◽  
Author(s):  
Morigen Morigen ◽  
Ingvild Flåtten ◽  
Kirsten Skarstad
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Binding Sites ◽  
Replication Initiation ◽  
Permissive Temperature ◽  
Initiator Protein ◽  
Dnaa Protein ◽  
Daughter Cells ◽  
Initiation Of Replication ◽  
Replication Initiator

In Escherichia coli inhibition of replication leads to a block of cell division. This checkpoint mechanism ensures that no cell divides without having two complete copies of the genome to pass on to the two daughter cells. The chromosomal datA site is a 1 kb region that contains binding sites for the DnaA replication initiator protein, and which contributes to the inactivation of DnaA. An excess of datA sites provided on plasmids has been found to lead to both a delay in initiation of replication and in cell division during exponential growth. Here we have investigated the effect of datA on the cell division block that occurs upon inhibition of replication initiation in a dnaC2 mutant. We found that this checkpoint mechanism was aided by the presence of datA. In cells where datA was deleted or an excess of DnaA was provided, cell division occurred in the absence of replication and anucleate cells were formed. This finding indicates that loss of datA and/or excess of DnaA protein promote cell division. This conclusion was supported by the finding that the lethality of the division-compromised mutants ftsZ84 and ftsI23 was suppressed by deletion of datA, at the lowest non-permissive temperature. We propose that the cell division block that occurs upon inhibition of DNA replication is, at least in part, due to a drop in the concentration of the ATP–DnaA protein.

scholarly journals Corepression of the P1 Addiction Operon by Phd and Doc

1998 ◽  
Vol 180 (23) ◽  
pp. 6342-6351 ◽  
Author(s):  
Roy Magnuson ◽  
Michael B. Yarmolinsky
Keyword(s):  
Fusion Protein ◽  
Binding Sites ◽  
Wild Type ◽  
Dna Complexes ◽  
Present Evidence ◽  
Cooperative Interactions ◽  
Regulatory Properties

ABSTRACT The P1 plasmid addiction operon encodes Doc, a toxin that kills plasmid-free segregants, and Phd, an unstable antidote that neutralizes the toxin. Additionally, these products repress transcription of the operon. The antidote binds to two adjacent sites in the promoter. Here we present evidence concerning the regulatory role of the toxin, which we studied with the aid of a mutation,docH66Y. The DocH66Y protein retained the regulatory properties of the wild-type protein, but not its toxicity. In vivo, DocH66Y enhanced repression by Phd but failed to affect repression in the absence of Phd, suggesting that DocH66Y contacts Phd. In vitro, a MalE-DocH66Y fusion protein was found to bind Phd. Binding of toxin to antidote may be the physical basis for the neutralization of toxin. DocH66Y failed to bind DNA in vitro yet enhanced the affinity, cooperativity, and specificity with which Phd bound the operator. Although DocH66Y enhanced the binding of Phd to two adjacent Phd-binding sites, DocH66Y had relatively little effect on the binding of Phd to a single Phd-binding site, indicating that DocH66Y mediates cooperative interactions between adjacent Phd-binding sites. Several electrophoretically distinct protein-DNA complexes were observed with different amounts of DocH66Y relative to Phd. Maximal repression and specificity of DNA binding were observed with subsaturating amounts of DocH66Y relative to Phd. Analogous antidote-toxin pairs appear to have similar autoregulatory circuits. Autoregulation, by dampening fluctuations in the levels of toxin and antidote, may prevent the inappropriate activation of the toxin.

scholarly journals Unilateral Cleavage Furrows in Multinucleate Cells

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1493 ◽  
Author(s):  
Julia Bindl ◽  
Eszter Sarolta Molnar ◽  
Mary Ecke ◽  
Jana Prassler ◽  
Annette Müller-Taubenberger ◽  
...  
Keyword(s):  
Actin Filament ◽  
Average Velocity ◽  
Electric Pulse ◽  
Myosin Ii ◽  
Cross Linking ◽  
Wild Type ◽  
Initial Direction ◽  
Multinucleate Cells ◽  
Daughter Cells

Multinucleate cells can be produced in Dictyostelium by electric pulse-induced fusion. In these cells, unilateral cleavage furrows are formed at spaces between areas that are controlled by aster microtubules. A peculiarity of unilateral cleavage furrows is their propensity to join laterally with other furrows into rings to form constrictions. This means cytokinesis is biphasic in multinucleate cells, the final abscission of daughter cells being independent of the initial direction of furrow progression. Myosin-II and the actin filament cross-linking protein cortexillin accumulate in unilateral furrows, as they do in the normal cleavage furrows of mononucleate cells. In a myosin-II-null background, multinucleate or mononucleate cells were produced by cultivation either in suspension or on an adhesive substrate. Myosin-II is not essential for cytokinesis either in mononucleate or in multinucleate cells but stabilizes and confines the position of the cleavage furrows. In fused wild-type cells, unilateral furrows ingress with an average velocity of 1.7 µm × min−1, with no appreciable decrease of velocity in the course of ingression. In multinucleate myosin-II-null cells, some of the furrows stop growing, thus leaving space for the extensive broadening of the few remaining furrows.

Three-dimensional in vivo analysis of Dictyostelium mounds reveals directional sorting of prestalk cells and defines a role for the myosin II regulatory light chain in prestalk cell sorting and tip protrusion

Development ◽  
2000 ◽  
Vol 127 (12) ◽  
pp. 2715-2728 ◽  
Author(s):  
P.A. Clow ◽  
T. Chen ◽  
R.L. Chisholm ◽  
J.G. McNally
Keyword(s):  
Long Range ◽  
Light Chain ◽  
Cell Sorting ◽  
Myosin Ii ◽  
Regulatory Light Chain ◽  
Upward Movement ◽  
Wild Type ◽  
Vertical Column ◽  
Initial Cluster

During cell sorting in Dictyostelium, we observed that GFP-tagged prestalk cells (ecmAO-expressing cells) moved independently and directionally to form a cluster. This is consistent with a chemotaxis model for cell sorting (and not differential adhesion) in which a long-range signal attracts many of the prestalk cells to the site of cluster formation. Surprisingly, the ecmAO prestalk cluster that we observed was initially found at a random location within the mound of this Ax3 strain, defining an intermediate sorting stage not widely reported in Dictyostelium. The cluster then moved en masse to the top of the mound to produce the classic, apical pattern of ecmAO prestalk cells. Migration of the cluster was also directional, suggesting the presence of another long-range guidance cue. Once at the mound apex, the cluster continued moving upward leading to protrusion of the mound's tip. To investigate the role of the cluster in tip protrusion, we examined ecmAO prestalk-cell sorting in a myosin II regulatory light chain (RLC) null in which tips fail to form. In RLC-null mounds, ecmAO prestalk cells formed an initial cluster that began to move to the mound apex, but then arrested as a vertical column that extended from the mound's apex to its base. Mixing experiments with wild-type cells demonstrated that the RLC-null ecmAO prestalk-cell defect is cell autonomous. These observations define a specific mechanism for myosin's function in tip formation, namely a mechanical role in the upward movement of the ecmAO prestalk cluster. The wild-type data demonstrate that cell sorting can occur in two steps, suggesting that, in this Ax3 strain, spatially and temporally distinct cues may guide prestalk cells first to an initial cluster and then later to the tip.

Role of myosin II activity and the regulation of myosin light chain phosphorylation in astrocytomas

2007 ◽  
Vol 65 (1) ◽  
pp. 12-24 ◽  
Author(s):  
Bodour Salhia ◽  
Jeong Hyun Hwang ◽  
Christian A. Smith ◽  
Mitsutoshi Nakada ◽  
Fiona Rutka ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Myosin Ii ◽  
Myosin Light Chain Phosphorylation
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