Cell Biol-Cell Cycle regulation

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Cell Biol-Cell Cycle regulation Powered By Docstoc
					                     Physiology of Animal Cells
  Topic: Regulating the Eukaryotic Cell Cycle (Chapter 21)
 Overview of Cell Cycle and Its Control
 An

Biochemical Studies of Oocytes, Eggs and Early Embryos

Genetic Studies of S. Pombe.

Molecular Mechanisms for Regulating Mitotic Events

Genetic Studies of S. cerevisiae

Cell Cycle Control in Mammalian Cells

Checkpoints in Cell Cycle Regulation

Meiosis: A Special Type of Cell Division

                  Instructor: 陳洵一, Chen Shuen-Ei




                     An Overview of Cell Cycle and Its Control
                                                   Cells in G0 phase, may
Onset of mitosis until
                                                   1) Exit the cell cycle
the end (2 daughter
                                                      forever and go into
cells are completely
                                                      differentiation program,
separated)
                                                      (Most of adult cells are
                                                      fully differentiated, no
                                                      proliferation capacity)
                                                   2) Re-enter the cycle,
                                                    3) Stay in a resting state
                                                        for signal stimulation
                                                        (adult stem cells are in
                                                        the G0 phase waiting
                                                        for signal stimulation)
                                                   The phase after mitosis
When the sister
                                                   completion until DNA
chromatids can
                                                   synthesis for replication
be observed
                                                   (most of mRNAs and
                                                   proteins are synthesized
                                                   in the phase).




                                                                                   1
An Overview of Cell Cycle and Its Control
                             Cyclins as the regulatory
                             subunit and CDKs (cyclin-
                             dependent kinases) as the
                             catalytic subunit for
                             heterodimmers to regulate
                             cell cycle




An Overview of Cell Cycle and Its Control

                                 Cells in G1 phase have
                                 intact nucleus
                                 Chromosomes in G1
                                 phase are not
                                 condensed and so
                                 individual chromosomes
                                 are indistinguishable



                             Fusion with M phase cells
                             causes condensed
                             chromatids and retract of
                             nuclear membrane into ER
                             in G1 cells→ factors from M
                             phase cells




                                                           2
            An Overview of Cell Cycle and Its Control
                                      Vectors containing many genomic
                                      DNA fragments of wild-type yeasts


                                                    Sequencing then
                                                    compared with
                                                    human genomic DNA
                                                    to identify human
                                                    homologous genes




 mutant cells can only
be grown at 25  C




            An Overview of Cell Cycle and Its Control




     MPF (mitosis-promoting factor) is commonly referred as a
     heterodimer composed of a mitotic cyclin and CDK.




                                                                          3
           Biochemical Studies of Oocytes, Eggs and Early Embryos
                When sexual
    After birth maturation




DNA replication and     Meiosis
sister chromatids       resumes
have been
completed




           Biochemical Studies of Oocytes, Eggs and Early Embryos




                                                                    4
         Biochemical Studies of Oocytes, Eggs and Early Embryos




         Biochemical Studies of Oocytes, Eggs and Early Embryos
  Early events such as chromosome
  condensation and nuclear envelop disassembly   Degrading all mRNAs




                                                 Cyclin B (the major factor
                                                 of MRFs in Xenopus)
                                                 needs to be degraded for
                                                 complete mitosis

Late events such as chromosome further
condensation and nuclear envelop assembly




                                                                              5
        Biochemical Studies of Oocytes, Eggs and Early Embryos

                                                APC (anaphase-promoting
                                                complex) is a ubiquitin ligase
                                                that transfers ubiquitin
                                                polymers (tags) to the target
                                                proteins for degradation




                                              Binding of APC to active Cdh1 (an
                                              APC specificity factor) leads to
                                              activated APC to ubiquitinate
                                              MRF for proteasomal degradation

Cdc14                                    Ubiquitin attached to the region
dephosphorylate                          containing 9 aa sequence called
cdh1                                     destruction box in the N-terminal




Ubiquitin System in Protein
Degradation

 E1: Ub-activating proteins
 E2: Ub-carriers (also called
 conjugating enzymes)
 E3: Ub-ligases




                                Proteosome
                                degradation




                                                                                  6
                             Genetic Studies S. Pombe.




Pre-mature mitosis (cells
don’t reach a normal size)



                                                    Timing of MRF
                                                    activation is important




                             Genetic Studies S. Pombe.
  In S. Pombe. (yeast)
                                      CDK-activating kinase




                 CAK cannot phosphorylate Y161 directly if y15
                 is not phosphorylated by Wee1




                                                                              7
         Molecular Mechanisms for Regulating Mitotic Events




Phosphorylation of lamins by MRF leads to de-
polymerization and disintegration of lamin networks
surrounding the inner nuclear membrane




         Molecular Mechanisms for Regulating Mitotic Events
                                    lamin




                                                              8
        Molecular Mechanisms for Regulating Mitotic Events




                                                               chromosomes
                                                               separation is not
                                                               different but no de-
                                                               condensation




                                                               Tubulins are still
                                                               visible (not
                                                               sequestered into
                                                               ER




        Molecular Mechanisms for Regulating Mitotic Events
Exogenous destruction box peptide competing with endogenous cyclin B
for ubiquitination destruction
                               Delayed chromosome separation and de-condensation
                                                   No chromosome separation



                                                                   From a, b, c
                                                                   experiments,→
                                                                   factors or
                                                                   proteins other
                                                                   than cyclin B are
                                                                   required for
                                                                   chromosome
                                                                   separaton




                                       continued




                                                                                       9
          Molecular Mechanisms for Regulating Mitotic Events




  Cdc20: an APC specificity factor (compared with Cdh1 that directs APC to MRFs)
  Separase: a protease
  Securin: a separase inhibitor
  SMC: a cohensin protein (microtubulins) linking two regions of the chromosomes
  SCC: a cohensin protein forming a joint of two SMC proteins from 2 different
  chromosomes




          Molecular Mechanisms for Regulating Mitotic Events




Re-phosphorylation of lamins leads to
polymerization and integration of lamin networks
surrounding the inner nuclear membrane
NPC: nuclear pore complex
                                                   Karyomeres fuse together




                                                                                   10
                         Genetic Studies of S. cerevisiae




                     (cdc28)




  SPF: S phase-promoting factor




          clin3
                         Genetic Studies of S. cerevisiae


Clib5,6




  1,G1 cyclin-CDK phosphorylates Sic1
  2, Sic1-P is polyubiquitinated by ubiquitin ligase (SCF)
      leading to proteosomal degradation
  3, activated S cyclin-CDK initiates DNA replication




                                                             11
                       Genetic Studies of S. cerevisiae

Clb 1, 2, 3, 4, 5,6:
B-type cyclins
(mitotic)




                       Genetic Studies of S. cerevisiae


                                      Mainly Clib1,2 in S. cerevisiae and
                                      cyclin A/B-CDK1 in vertebrates and B
                                      S cyclin-CDK is required for initiation of
                                      DNA replication mainly cyclin A/E-CDK2
                                      in vertebrates




                                                                                   12
                     Cell Cycle Control in Mammalian Cells

                                      BrdU: Bromo-
                                      deoxy-UTP; a
                                      fluorescent dye




                     Cell Cycle Control in Mammalian Cells
                                                             yeast




Usually for most in vitro cultured mammalian
cells, a cell cycle takes 24 h




                                                                     13
               Cell Cycle Control in Mammalian Cells




                                  G1 cyclins: cyclin D and E and CDK2, 4 and
All with a destruction box        6 (also called D-type cyclin)
sequence recognized by
APC                                 B type cyclins: cyclin A and B




                    Cell Cycle Control in Mammalian Cells


                                             Serum (containing growth
                                             factors) can initiate cell cycle


                                               When protein synthesis
                                               inhibitors are present, no protein
                                               syntheses from delayed-
                                               response genes →
                                               1)early response gene
                                               expressions lead to delayed-
                                               response gene expression
                                               2) delayed gene products can
                                               degrade early gene mRNA (and
                                               products)
                Early response genes such as c-fos , c-Jun, c-myc, that
                can induce many gene expressions including cyclins




                                                                                    14
                   Cell Cycle Control in Mammalian Cells
Mitogens such as
IGF, insulin and                                             Positive feedback
other growth factors                                         regulation
induce early
response gene
expressions first
then




                                             When anaphase is completed and
                                             cells enter G0 or G1 phase,
                                             phosphorylation of Rb drops rapidly,
                                             and cells wait for another cycle




                   Cell Cycle Control in Mammalian Cells



                                                       Cyclin D,E and
                                                       CDK4 and 6




  Cyclin A and B                                               Mainly
  and CDK1                                                     Cyclin A
                                                               and CDK 2




                                                                                    15
APC ubiquitinates
cyclinA/B-CDK1        Cell Cycle Control in Mammalian Cells
for degrdation han                                   Mitogens induces early-response
cell enter G1 phase                                  gene expressions and following
                                                     delay-response gene expressions
                                                     such as cyclin D-CDK/4/6

Cdc25C, a phosphatase,
can remove an inhibitory
phosphate from CDK1                                      Can phosphorylates Rb and
and leads to activation                                  as a result releases E2F that
of cyclinA-CDK2                                          in turn induces cyclinE-CDK2
                                                         expression and a further
                                                         positive feedback regulation
                                                         (see the previous slide)

                                                          INK (inhibitors of kinase)
                                                          such as p16 can bind to
   Cdc25A, a phosphatase, can
                                                          CDK4/6 to prevent the
   remove a inhibitory phosphate
                                                          interaction with cyclin D
   from CDK2 and leads to activation
   of cyclinA-CDK2                           Phosphorylation of p27 by cyclin E-CDK2
                                             targets p27 to ubiquitination by SCF.

   3 CDK inhibitory proteins (CIP):P21,   Phosphorylation of p21by Akt (akt is
   p27, and p57 can bind to cyclin D-     activated by mitogens) leads to release of
   CDK4/6 and cyclinA/E-CDK2 to inhibit   cyclinA/E-CDK2 (at T145) and an increase
   their assembly and activities          cyclinD-CDK4/6 activity (at S146) (p21 is
                                          still bound with cyclinD/CDK4/6)




                      Checkpoints in Cell Cycle Regulation

  Checkpoints: to minimize
  occurrence of mistake in
  cell cycle progression in
  several key events




                                                                                         16
                    Checkpoints in Cell Cycle Regulation
       Mitotic arrest
       deficient (MAD)
       proteins
ATM/R: ataxia telangiectasia
mutated and related protein
kinase




                    ATR: ATM and rad3
                    related kinase

 Chk: checkpoint
 kinase

            P53 is a transcription factor that can induce p21 expression.




                    Checkpoints in Cell Cycle Regulation
 1, DNA replication checkpoint: If DNA replication is not proceeded properly
 a) Activated ATR binds to the folks of replicating DNA to phosphorylate and
    activate chk1. Activated chk1 can phosphorylate cdc25c to inactivate its
    phosphatase activity.
 b) As a result, an inhibitory phosphate from CDK1 can not be removed by
    inactivated cdc25c and cyclinA/B-CDK1 remains inactivated and can not
    phosphorylate targets required for mitosis initiation

 2, Spindle assembly checkpoint: If Mad (originally bound to kinetochores) are
     not linked with microtubulins. It can be converted into a soluble free form to
     interact with and inhibit cdcd20 activity.
 a) If spindle assembly is not proceeded properly (no attachement of
    microtubulins to kinetochores and Mad), Mad are released to interact with
    and inhibit cdc20 activity.
 b) As a result, cdc20 can not direct APC to poly-ubiquitinate securin for
    degradation and following separase is not released to cut off the
    microtubulins linking chromosomes and cells can not enter anaphase. This
    process ensure a precise chromosome attached to spindle for next
    segregation.




                                                                                      17
                 Checkpoints in Cell Cycle Regulation
3, Chromosome segregation checkpoint: When chromosomes separation and
    cytokinesis are not completed properly. cdc14 is not released from
    nuclelous to activate cdh1 and following events (See Figure 21-10, slide 11)

4a, DNA damage checkpoint: When DNA damage can not be repaired
a) Activated ATM/R phosphorylate P53 to prevent p53 degradation and thus
   sustained p53 levels can induce p21 expression.
b) Increased P21 expression then bind to prevent assembly and activation of
   cyclin D-CDK4/6

4b,c, DNA damage checkpoint: When DNA damage can not be repaired
a) Similar to 4a, except the final target, cyclin E/A-CDK2 .
b) Activated ATM/R also phosphorylate cdc25a to result in
   polyubiquitination of cdc25a degradation. As a result, an inhibitory
   phosphate from CDK2 can not be removed, and cyclin E/A-CDK2 remain
   inactivated and can not phosphorylate targets required for entry into S or
   G2 phase

4d, DNA damage checkpoint:
Similar to 4a, except the final target, cyclin A/B-CDK1.




                  Meiosis: A Special Type of Cell Division




Meiosis I                           Meiosis II




                                                                                   18
                   Meiosis: A Special Type of Cell Division
               Scc1 only
               attached to the
               centric regions

1) Rec8 (a meiotic homolog of
mitotic Scc1, a cohensin protein)
attached to the regions alone the 2
replicated chromosomes
2) After cross over, synapsis is
formed between 2 paired arms of
homologous chromosomes
(parental). Rec8 is then cleaved by
separase at the arm regions but not
at the centromere regions




                   Meiosis: A Special Type of Cell Division




                                                     Association of kinetochores
                                                     with spindle microtubules
                                                     and cohensin degradation for
                                                     complete chromosome
                                                     separation depends on
                                                     factors that are associated
                                                     with the chromosomes but
                                                     not on he spindles and
                                                     cytosolic soluble factors




                                                                                    19