Lecture 32 and on - Cell Biology

regulation of actin cytoskeleton by G-proteins
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stages of the cell cyclecells can be sorted on the basis of differences in DNA contentasynchronous populationsynchronous populationcell cycle controlthis ensures the sequential occurrence of critical events control mechanism monitors internal and external conditions before triggering next stepdiscovery of components of control mechanismbiochemists devised an assay or readout using frog oocytes to know whether the cell cycle has been activated - poised to perform mitosis (arrested in G2) - can be used as a readout to test candidate proteins - synchronised cells, so egg extracts can be analyzed biochemically to discover cell-cycle associated molecules - discovered MPF geneticists discovered temperature-sensitive cell division cycle (cdc) mutantsbiochemistsbreak open cells and isolate proteins or chemicals to find something that controls the cell cyclegeneticistsbreak the cell cycle using mutations to discover genes involved in itmaturation-promoting factorhas ability to induce mitosis in resting oocytes purified MPF has two components: - kinase - mitotic cyclin complex of M-Cyclin and M-cyclin dependent kinase function is conserved from yeast to humanscyclinproteins whose concentration varies periodically (cycles) in coordination with specific phases of the cell cyclemitotic cyclinactivation of mitotic cdkabrupt rise in M-Cdk activity abrupt loss of M-Cdk activityabrupt rise in M-Cdk activityremoval of inhibitory phosphateabrupt loss in M-Cdk activityM-cyclin degradationAPCanaphase-promoting complex ubiquitylates cyclin in abrupt loss of M-Cdk activity induces M to G1 transitiondifferent Cdks for different phasesG1-Cdkcyclin-Cdk complex pairs with Cyclin D CDK partner: Cdk4, Cdk6cyclin Dthree in mammals: D1, D2, D3p27blocks G1 to S transitionG1 cell checkpointsdamaged DNA unfavorable extracellular environment p27 blocks G1 to S transitionS cell checkpointsdamaged or incompletely replicated DNAG2 cell checkpointsdamaged or incompletely replicated DNAM checkpointschromosome improperly attached to mitotic spindle APC induces M to G1 transitionG0 phasephase in the cell cycle, when the cell is in quiescent state extended G1 phase cell is neither dividing nor preparing to dividecell cycle withdrawalmany differentiated cell types withdraw into G0 in G0 phase, Cdks and cyclins degradedcell cycle control mechanismsinhibition of activating phosphatase Cdc25 blocks entry to mitosis inhibition of APC activation delays exit from mitosis Cdk inhibitors block entry to S phaseinhibition of Cdc25inhibition of activating phosphate blocks entry into mitosisinhibition of APC activationdelays exit from mitosis no ubiquitylation of cyclin by APCCdk inhibitorsblock entry to S phasecell cycle initiation and progressNO Cdk activity at the end of M-phase G1-Cdk-cyclins for progress through G1 phase G1/S-Cdk cyclins and S-Cdk cyclins for entry into S phase - these complexes are inactive in G1 due to inhibitory proteins, resulting in G1 arrest mitogenic signals and other factors will relieve inhibition (G1 arrest) and cells will exit G1 M-Cdk-cyclins for entry into M phase. these complexes are inactive in G2early frog embryo cellscell type with a 30 minute cell-cycle time no G1 phasearrest at G1cell must grow (increase mass) environment must be favorable DNA must be free of mutations p53 protein & Rb protein multiple conditions must be satisfactory will be relieved only when damaged DNA has been repaired and mitogens are presentdefects in DNA repair machineryBRCA1 and BRCA2 tumor suppressors x-rays cause DNA damage, active p53 blocks cell divisionp53blocks cell division when active activated by protein kinases that phosphorylate this in absence of DNA damage, this protein is degraeded in proteasomes binds to regulatory region of p21 gene leading to G1 arrest tumor suppressor reason: DNA MUST BE FREE OF MUTATIONSconsequences of mutations that inactivate p53cells will divide unchecked cells will accumulate mutations high probability of cancer p53 is a tumor suppressorRb proteinreason: ENVIRONMENT MUST BE FAVORABLE retinoblastoma arrests G1 phase tumor suppressorunfavorable environmentno growth factors or mitogens active Rb blocks cell division in G1 arrestmitogenan agent that triggers mitosisconsequences of mutations that inactivate Rbcells will lose mitogen dependence cells will divide unchecked high probability of cancer Rb is a tumor suppressoreffects of constitutive signalingpathway ON without mitogen activating mutations in RAS oncogeneG1 arrest mechanismsp53 - deals with damaged DNA Rb protein - deals with unfavorable extracellular environment inactivated tumor sppressor genes (p53, Rb, BRCA1, BRCA2) contribute to cancer activated proto-oncogenes (eg., RAS) contribute to cancerpositive feedbackfeedback that tends to magnify a process or increase its outputnegative feedbacka mechanism of response in which a stimulus initiates reactions that reduce the stimulusmitosisinterphaseconsists of G1, S, G2 the cell increases in size DNA of the chromosomes is replicated and the chromosome is duplicatedprophasereplicated chromosomes, each consisting of two closely associated sister chromatids, condense outside the nucleus, the mitotic spindle assembles between the two centrosomes, which have begun to move apartprometaphasestarts abruptly with the breakdown of the nuclear envelope chromosomes can now attach to spindle microtubules via their kinetochores and undergo active movementmetaphasechromosomes are aligned at the equator of the spindle, midway between the spindle pores paired kinetochore microtubules on each chromosome attach to opposite poles of the spindleanaphasesister chromatids separate synchronously each chromatid is pulled slowly toward the spindle pole it is attached to kinetochore microtubules get shorter and the spindle poles also move apart, both contributing to chromosome segregationtelophasetwo sets of chromosomes arrive at the poles of the spindle new nuclear envelope reassembles around each set completing the formation of two nuclei and marking the end of mitosis division of the cytoplasm begins with the assembly of the contractile ringcytokinesiscytoplasm is divided in two by a contractile ring of actin and myosin filaments, which pinches in the cell to create two daughters, each with one nucleusDNA binding proteinscondensins and cohesinscondensinshelp condense chromosomes during prophase (triggered by M-Cdk)cohesinskeep sister chromatids together during metaphasecytoskeleton rolesmicrotubules - control chromosome movement actin filaments - control cytokinesis