11 research outputs found
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Bleb-driven chemotaxis in Dictyostelium discoideum
Migrating cells have two basic ways of extending their leading edge: by dendritic actin polymerization beneath the membrane, or by fluid pressure, which produces blebs. Most cells are believed to move using actin-driven projections, but in more physiological conditions, blebbing motility is also apparent. It has been shown that certain cells even can switch between these two modes of motility, although it is not known how this switch is triggered. Besides, it is unclear whether blebbing can be regulated by chemotactic stimuli, and generally, how blebbing is controlled in the cell.
In this study I employed a popular model organism â Dictyostelium discoideum â to investigate the role of blebbing in chemotaxis. Here I confirm that in standard conditions Dictyostelium cells move by a combination of F-actin-driven protrusions and blebs. Blebbing is characterized by the rapid projection of hemispherical patches of plasma membrane at 2-4 times the speed of an actin-driven projection, and leaves transient scars of F-actin marking the original cortex in the base of blebs. I demonstrate that Dictyostelium cells can adjust their mode of movement according to the conditions: in a resistive environment they switch almost entirely to âbleb modeâ.
I show that in chemotaxing cells, blebs are mainly restricted to the leading edge, and they often lead the way when a cell is forced to re-orientate. Bleb location appears to be controlled directly by chemotactic gradients. To investigate how chemoattractant induces blebbing, I have screened signal transduction mutants for altered blebbing. I have found that blebbing is unaffected in many chemotactic mutants, but unexpectedly depends on PI3-kinases and two downstream PIP3-binding proteins of unknown function â PhdA and CRAC.
I conclude that Dictyostelium cells move using a hybrid motor in which hydrostatic pressure-driven bleb formation is as important as F-actin-driven membrane extension, and that cells can change the balance between modes as required. I propose that blebbing motility of Dictyostelium cells is a direct response to mechanical resistance of environment. More generally, bleb-driven motility may be a ââhigh-forceâ mode of movement that is suited to penetrating tissues. Blebs are chemotactic and their induction may involve branches of the chemotactic signal transduction pathway distinct from F-actin regulation.This work was supported by the Herchel Smith Fellowshi
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Cyclin D-Cdk4,6 Drives Cell-Cycle Progression via the Retinoblastoma Protein's C-Terminal Helix.
The cyclin-dependent kinases Cdk4 and Cdk6 form complexes with D-type cyclins to drive cell proliferation. A well-known target of cyclin D-Cdk4,6 is the retinoblastoma protein Rb, which inhibits cell-cycle progression until its inactivation by phosphorylation. However, the role of Rb phosphorylation by cyclin D-Cdk4,6 in cell-cycle progression is unclear because Rb can be phosphorylated by other cyclin-Cdks, and cyclin D-Cdk4,6 has other targets involved in cell division. Here, we show that cyclin D-Cdk4,6 docks one side of an alpha-helix in the Rb C terminus, which is not recognized by cyclins E, A, and B. This helix-based docking mechanism is shared by the p107 and p130 Rb-family members across metazoans. Mutation of the Rb C-terminal helix prevents its phosphorylation, promotes G1 arrest, and enhances Rb's tumor suppressive function. Our work conclusively demonstrates that the cyclin D-Rb interaction drives cell division and expands the diversity of known cyclin-based protein docking mechanisms
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Cyclin D-Cdk4,6 Drives Cell-Cycle Progression via the Retinoblastoma Proteins C-Terminal Helix.
The cyclin-dependent kinases Cdk4 and Cdk6 form complexes with D-type cyclins to drive cell proliferation. A well-known target of cyclin D-Cdk4,6 is the retinoblastoma protein Rb, which inhibits cell-cycle progression until its inactivation by phosphorylation. However, the role of Rb phosphorylation by cyclin D-Cdk4,6 in cell-cycle progression is unclear because Rb can be phosphorylated by other cyclin-Cdks, and cyclin D-Cdk4,6 has other targets involved in cell division. Here, we show that cyclin D-Cdk4,6 docks one side of an alpha-helix in the Rb C terminus, which is not recognized by cyclins E, A, and B. This helix-based docking mechanism is shared by the p107 and p130 Rb-family members across metazoans. Mutation of the Rb C-terminal helix prevents its phosphorylation, promotes G1 arrest, and enhances Rbs tumor suppressive function. Our work conclusively demonstrates that the cyclin D-Rb interaction drives cell division and expands the diversity of known cyclin-based protein docking mechanisms
Recommended from our members
Cyclin D-Cdk4,6 Drives Cell-Cycle Progression via the Retinoblastoma Protein's C-Terminal Helix.
The cyclin-dependent kinases Cdk4 and Cdk6 form complexes with D-type cyclins to drive cell proliferation. A well-known target of cyclin D-Cdk4,6 is the retinoblastoma protein Rb, which inhibits cell-cycle progression until its inactivation by phosphorylation. However, the role of Rb phosphorylation by cyclin D-Cdk4,6 in cell-cycle progression is unclear because Rb can be phosphorylated by other cyclin-Cdks, and cyclin D-Cdk4,6 has other targets involved in cell division. Here, we show that cyclin D-Cdk4,6 docks one side of an alpha-helix in the Rb C terminus, which is not recognized by cyclins E, A, and B. This helix-based docking mechanism is shared by the p107 and p130 Rb-family members across metazoans. Mutation of the Rb C-terminal helix prevents its phosphorylation, promotes G1 arrest, and enhances Rb's tumor suppressive function. Our work conclusively demonstrates that the cyclin D-Rb interaction drives cell division and expands the diversity of known cyclin-based protein docking mechanisms
Mass spectrometry and biochemical analysis of RNA polymerase II: Targeting by protein phosphatase-1
Transcription of eukaryotic genes is regulated by phosphorylation of serine residues of heptapeptide repeats of the carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII). We previously reported that protein phosphatase-1 (PP1) dephosphorylates RNAPII CTD in vitro and inhibition of nuclear PP1-blocked viral transcription. In this article, we analyzed the targeting of RNAPII by PP1 using biochemical and mass spectrometry analysis of RNAPII-associated regulatory subunits of PP1. Immunoblotting showed that PP1 co-elutes with RNAPII. Mass spectrometry approach showed the presence of U2 snRNP. Co-immunoprecipitation analysis points to NIPP1 and PNUTS as candidate regulatory subunits. Because NIPP1 was previously shown to target PP1 to U2 snRNP, we analyzed the effect of NIPP1 on RNAPII phosphorylation in cultured cells. Expression of mutant NIPP1 promoted RNAPII phosphorylation suggesting that the deregulation of cellular NIPP1/PP1 holoenzyme affects RNAPII phosphorylation and pointing to NIPP1 as a potential regulatory factor in RNAPII-mediated transcription. © 2010 Springer Science+Business Media, LLC