BIOLOGY 2B03 Lecture Notes - Lecture 10: Nuclear Lamina, Lamin, Spindle Apparatus

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27 Apr 2016
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2B03- Cell Cycle Dynamics and Checkpoints
Entry into Mitosis
-Active MPF- heterodimer of mitotic cyclin and CDk- allow changers to occur when cells enter prophase
-At the end of mitotic, all the process are reversed by inactivating MPF by ubiquitin-mediated degradation of Cyclin B
Active MPF: Prophase Events
-Initiated processes by active MPF phosphorylating target proteins:
oMitotic spindle formation: phosphorylate microtubule-associated proteins that promote microtubule instability and
centrosome separation
oCondensation of chromosome: phosphorylate condensins and histone proteins
oPreparation of sister chromatid separation: phosphorylation of cohesins, contributing to condensation
oNuclear envelope breakdown (NEB): phosphorylate nuclear lamins
oBreakdown of Golgi and ER: phosphorylate GM130
-Early prophase: centrosomes not separate, nuclear enveloped attached, chromosomes are starting to condnse
-End of prophase/enter metaphase: centrosomes separated and organized into bipolar spindle, NEB and chromosoems fully
condensed and bound to mitotic spindle
Inactivation of MPF Kinase; Telophase Events
-MPF is deactivated and phosphatases dephosphorylated the target proteins
-In anaphase: sister chromatid separation occurs
-End of telophase: nuclear envelope reforms around segregated set of daughter chromosomes, chromosomes decondense,
mitotic spindle disassembled and Glolgi and ER membranes reassemble
Chromosome Condensation
-Chromatin is highly dispersed in interphase but during mitosis, they begin to
condense and become distinguishable
-In chromosome:
oCentromeres are closely attached by cohensin complexes
oArms are separated to form an X shape
-Codensins, cohesins, histones (H1 and H3)and topoisomerase are all
phosphorylated to achieved chromosome condensation
Histones:
-Chromosomes are wrapped around binding proteins called histones
oHistone H3 is pat of the octamer and forms the core of the nucleosome
oH1 is the linker between neighbouring
nucleosomes
-Nucleosomes are packed close together during
chromosome condensation
-Aurora B, kinase, phosphorylates H1 and H
during chromosome condensation
oExperiment performed to see this, and
antibodies only bind to the
phosphorylated form of the protein, not non-phosphorlyated
Cohesins:
-Cohesins form the cohesion complex needed to hold sister chromatids
together after replication until anaphase
-To release cohesins:
oIn phosphase, many cohesins are released at the chromosome
arms, creating the X shape of the chromosome
Cohensins at the centromere are protected by
phosphorylation, in part by phosphatases
oIn metaphase, the X shape is observed
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oIn anaphase, the cohesion are targeted by CyclinB-CDK and Aurora B for dissociation
Cohesion at the centromere are targeted = chromatid separation
Condensins:
-During prophase, when the cohesins is lost on the arm, codensin proteins being to
assemble into a complex
-Condensin is phosphorylated by CyclinB-CDK
oMultiple MPF phosphorylated ites are found on the C-terminal end of
Xenopus codensins, called XCAP-D2
-During chromosome condensation, the cohesions will compact the sister chromatids
together, while the codensin will compact regions on the same DNA molecule
together
Chromosome Decondensation
-During telophase, the condensation of chromosomes in each daughter cell decondenses
-Condensation is needed for transcription to occur in interphase (G1)
-In absence of CyclinB/CDK, and presense of phosphatase, histones, condensins and cohesion are dephosphorylated
oCauses condensins to be removed and DNA remodels around histones
NEB
-In fluoresecences, the inner nuclear membrane’s lamin A protein is highlighted
-Interphase: envelope intact; early prometaphase; starting to break down; metaphase: completely gone- fragmented into small
vesicles and distributed in the cytosol
Lamins
-There is an other layer (ONM) that is continuous with RER
and inner membrane (INM) that is a scaffold of
intermediate filaments called nuclear lamina
-Nuclear pore complexes (NPC) found throughout both
layers and allow for transport in/out of the nucleus
-Interphase: chromosomal proteins interact with nuclear
lamina to anchor chromatin via mechanism of regulating
gene expression
-Nuclear lamina made up of proteins, that polymerize to form a mesh
network
oLamina A, B and C
-Prophase: 3 lamins are phosphorylated at specific serine resides by
CyclinB/CDK
oStarts disassembly of nuclear lamina by breaking down the lamina
tetramer
Lamina B stays associated with nuclear membrane
Lamin A and C dimers are soluble and disperse in cytosol
oINM starts to fragment
Necessary Phosphorylation
Experiment 1: In hamster cells, researchers added human wildtype Lamina A protein (similar between species, so lamina A was
incorporated into nuclear lamina
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-Interphase: intact nuclear lamina (therefore intact nuclear envelope)
-Prophase: Nuclear lamins tarts breaking down with apparent
involutions and chromosomes start to condense, lamina diffuse into
cytosol
-Metaphase: lamina A is no longer organised into a spherical ring and
chromosomes are fully compacted
Experiment 2: Used a variant of the human lamina, that produced lamina A that
cannot be phosphorylated- serine replaced with alanine.
-Interphase: same
-Prophase: chromosome condense, the involutions of the nuclear
lamina are visible
oLumina B and C can still be phosphorylated so these initial
actinons still occur
oLamina A does not diffuse into the cytosol
Remains in the unphosphorylated teramenres
-Metaphase: chromosomes are condensed but there’s still a ring around the chromosomes
Thus, phosphorylated of lamina A is necessary for nuclear lamina disassembly and NEB
Reassembly of the Nuclear Envelope
-Inactivation of Cyclin B/CDK kinase and activity of phosphatase, Cdc14, allows for dephosphorylation fo lamina A, B and C
-Lamisn reform tetramers and filaments to reform nuclear lamina
oLamina B stays associated with small vesciles formed from the nuclear lamina after NEB
So when the nuclear lamina reforms, lamina B brings the vesicles which can fuse with the surface of the
nuclear lamina to form the INM
-Early and late anaphase: nuclear envelope is dispersed in cytosol
-Telphase: nuclear envelope is seen forming around decondensing chromosomes
-Also, chromosomal proteins that interact the nuclear lamina and anchor the chromosomes are also phosphorylayed in anaphase
oPhosphorylation cause chromosomes to dissociate from nuclear lamina
oDephosphorylated allows the nuclear lamina to assemble around the chromosomes
-Nuclear pore complex proteins are phosphorylated at NEB, and it’s dephosphorylated at telophase as the nuclear envelope
reforms
Karyomere Fusion
Experiment: In cell-free system, Xenopus sperm was added to egg extracts
-Chromosomal proteins allowed vescicles to form around individual chromosoems
-Vesicles began to forma membrane around each chromosome, called a karyomere
oRegulated by Rab proteins;
-Karyomeres around each chromosome fuse together to form a complete nuclear envelope
oRab-like proteins mediate fusion of karymere
Golgi Fragmentation
-At the end of mitosis, organelles are reorganized into daughter cells; ~ equal separation
-Dictoyokinesis: Golgi division
-Before cytokinesis, clusters of Golgi fragments and vescicles form around spindle pore to ensure that a proportion of the
fragmented organelles go to each daughter cell
-One Golgi protein needs to be phosphorylated for fragmentation
-GM130 is phosphorylated by Cyclin B/CDK in mitosis as Golgi fragmentation begins
-Proteins stays phosphorylated until telophase, when the Golgi assembles in each daughter cell
oDephosphorylated of GM130 correlates with inactivation of Cyclin B/CDK telophase
Mitochondria Fragmentation
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