Lecture 7Chromosome Structure and Function in MitosisOutline:Chromosomes in MitosisCentromeres and KinetochoresChromosomes, Kinetochores and the SpindlePaper:ACTIVATION INACTIVATIONgain epigenetic marklose epigenetic markprimary sequence is not sufficient (dicentrics)‘marked’ DNA/chromatinEpigenetic Model for CEN Identitynon-centromeric sequence can acquire and propagate centromere function (neocentromeres)H4CENP-AdimerH2A/BH3 dimerCENP-A: An epigenetic mark for centromere identity?H3-like histone variantHistone H3C. elegans HCP-3S. cerev. CSE4S. pombe Cnp1Human CENP-AD. melano. CIDCENP-As are Conserved H3-like ProteinsH3 CORETAILconserved CEN-specific histone, but highly divergentrequired for kinetochore formation, segregation, mitotic progression, in flies,worms, mammals, yeastshigh (est?) in kinetochore assembly and function pathwayacting like a key mark for CEN identityCENP-A: Functional Foundation for the Kinetochoreabsence of CENP-A/kinetochore leads to chromosome loss/aneuploidyMitotic Defects caused by CENP-A DepletionH2B-RFP CENP-A-GFPCENP-A RNAipoor condensation / congressionno anaphase movementcytokinesis cuts chromosome massextended chromatin fibersCENP-AH3MERGE0.3-1.5 Mbhuman and flyCOREH4-AcBlocks of H3 and CENP-A Nucleosomes are Interspersed in CEN ChromatinTextH3 diMeK9(heterochromatin)+ -H3 H3 CENP-AHuman and flyH3 diMeK4(euchromatin)- +Are there other epigenetic ‘marks’ at centromeres besides CENP-A?CENs are embedded in heterochromatin- but are they modified like heterochromatin ?distinct from ‘classical’ Euchromatin & HeterochromatinCEN, EUCH, & HET Contain Distinct ModificationsHumanHumanDrosophilaCENP-ACENP-COUTERin the right place to nucleate the kinetochoreForms HIgher-Order ‘Cylindrical’ Structure in Mitotic Chromosomesinner and outer kinetochore proteins ‘wrapped’ around CENP-A cylinderSpiral/Loop Model for 3D Structure of CEN Chromatin‘presentation’ of CEN chromatin for attracting kinetochore proteinslittle or no H3 in cylinders, no mixing in mononucleosomeshow reconcile 3D exclusion and 2D interspersion of H3?Centromere region contains distinct domains‘stacking’ of similar nucleosomes ? HET H3, CEN H3, CENP-A- role of distinct modifications and flanking HET: 3D structure ? concentrate cohesin? epigenetic propagation of CEN identity (w/CENP-A)?CENP-A and Centromeric Chromatin:but how is CENP-A loaded only at centromeres ?unique chromatin composition and organization provides ample components for epigenetic regulationHow does the kinetochore function during mitosis ?Functional & Structural Foundation for the KinetochoreprometaphaseanaphasemetaphaseSpindle Anatomyhow is the spindle formed?Review: microtubule dynamicsundergo dynamic instabilitymicrotubules grow from MTOC (centrosome)parameters: growth rate (Vg) shrinkage rate (Vs) catastrophe frequency (fcat) rescue frequency (fres)modified by many cellular factorsMitotic microtubule dynamics and spindle assemblyConcept:Factors with opposing activities modulate MT dynamics1) global changes in MT dynamics2) local modulation of MT dynamics: chromosome attachment and congressionConcept:chromosome movements are linked to MT dynamics and motor proteins 3) organization into a spindleConcept:Motors with opposing activities arrange MTsStablemicrotubulesQuestions:1) what MT parameters change?2) what generates this global change?DynamicmicrotubulesPhotobleaching studies: interphase MTs t1/2 ~ 5 min mitotic MTs t1/2 ~ 0.5 minApproach:use a system in which dynamics are easily measuredidentify and deplete different factorsreconstitute dynamics with pure tubulin and factorsXenopus egg extractsprimary parameter affected: fcatastrophe increasesPossible regulatory mechanisms: stabilizing MAPs inactivated and/or catastrophe factors activatedOpposing factors identified: XMAP215 & XKCM1MTspolymerizedfromcentrosomesTournebize et al., Nature Cell Biol. 2, 13 (2000)XMAP215 stabilizes MTs in interphase and mitosisModel: XKCM1 constitutively active XMAP215 activity varies during cell cycleother cellular factors must also contribute to MT dynamicsConclusions:SpindleStablemicrotubulesDynamicmicrotubulesmicrotubuleslocally stabilizedattraction of MTs to chromosomesattachments to kinetochores and regulation by motorsbalance of motor-driven and MT assembly / disassembly forcesXMAP215yellow = XMAP215green = tubulinblue = DNAXMAP215 is on spindle but not astral microtubulestubulinMAP distribution is locally regulatedget stabilization of spindle but not astral microtubulesChromosome Attachment and Making a Bipolar Spindle2) search and capture - kinetochores capture MTs2 models:1) biochemical signal on chromosomes - diffusible molecules stabilize MTsnot mutually exclusiveMT growth toward chromosomes due to diffusible factors, independent of kinetochoresBiochemical signals on chromosomesbest evidence:micromanipulation in insect spermatocytes (Nicklas lab) removal of chromosomes decreases MT massXenopus: plasmid DNA coated beads chromatin enzyme generates signalRanGTP pathwaynuclear import/exportchromatin boundChromatin - RanGTP pathwayOther APA (aster-promoting activities):TPX2Rae1Exchange factor RCC1bound to chromatinChromatin - RanGTP recruitment of MTsSearch and capture: Kinetochores and MTslarge- Mbs of DNA, kinetochore binds ~30 MTs~80-90 proteinsorganized as subcomplexes with different and overlappingfunctionsThe Metazoan KinetochoreMT plus end bindingMT attachment and movementcheckpoint signalingCENP-E, dynein = motor proteinsMarginally more simple in yeast small- 125 bp DNA, kinetochore binds single MT~70 proteinsmany homologous to metazoan kinetochore proteinssimilar functional complexescheckpoint signalingMT attachment and movementSearch and capturedynamic MTs act as searching devicesonce captured by kinetochore, MT is stabilized1) video recordings of Newt lung cells2) physical interaction between MTs and kinetochores in vitrobest evidence:kinetochore capture in Newt lung cellSearch and captureprometaphasemetaphaseHow do chromosomes gain bipolar attachments and congress ?Sister chromatid pairs move to poles, congress to plateChromosome congressionMicrotubules attach to kinetochores and chromosomes oscillateKinetochore MTs (K-MTs) polymerize/depolymerize at their plus ends kinetochore stays attached!!! Chromosome arms are pushed to the metaphase platepolepoleMT assembly/disassembly promotes bipolar attachment and congressionHow is kinetochore movement