How are chromatids separated in anaphase? DIFFERENT FORCES LEAD TO SEPARATION OF SISTER CHROMATIDS: IN ANAPHASE A (ON THE LEFT) SHORTENING OF KINETOCORE MICROTUBULES IS THE MAIN FORCE. IN ANAPHASE B (ON THE RIGHT) MOLECOLAR MOTORS CAUSE SLIDING OF INTERPOLAR MICROTUBULES WHILE MEMBRANE BOUND DYNEINS PULL ASTRAL MICROTUBULES..

Tubulin movement in metaphase and anaphase IN METAPHASE, A DYNAMIC EQUILIBRIUM OF KINETOCORE MICROTUBULES IS MAINTAINED DUE TO TREADMILLING. IN ANAPHASE A RAPID DEPOLYMERIZATION AT THE KINETOCORE MICROTUBULE + END OCCURS. THESE ANALYSES ARE MADE BY FLUORESCENT TUBULIN INJECTION

Kinetocore microtubules rapidly depolymerize at the plus end DEPOLYMERIZATION OF MICROTUBULE +END AT THE LEVEL OF KINETOCORE REPRESENTS A MAJOR FORCE THAT PULLS EACH CHROMOSOME TO THE POLE DURING ANAPHASE. ANAPHASE

Motor proteins pull the poles far apart IN ANAPHASE B MOTOR PROTEINS OF KINESINE FAMILY CAUSE THE SLIDING OF INTERPOLAR MICROTUBULES IN THE OVERLAP REGION (AT CENTER). MOTOR PROTEINS OF DYNEIN TYPE EXCERT A FORCE OF ASTRAL MICROTUBULES PULLING CENTROSOMES TOWARD THE CELL PERIPHERY.

Many motor proteins act in mitosis

Sister chromatid separation at anaphase is synchronous THE MOVIE SHOWS AN ANIMAL CELL OBSERVED BY DIC (DIFFERENTIAL INTERFERENCE CONTRAST) DURING MITOSIS. CHROMOSOMES ALIGN AT THE EQUATOR OF THE MITOTIC SPINDLE AND THEN SEPARATION OF SISTER CHROMATIDS OCCURS. NOTE THE SYNCHRONY OF SEPARATION.

Sister chromatid separation at anaphase is synchronous THE MOVIE SHOWS A PLANT CELL OBSERVED BY PHASE CONTRAST DURING MITOSIS. CHROMOSOMES ALIGN AT THE EQUATOR OF THE MITOTIC SPINDLE AND THEN SEPARATION OF SISTER CHROMATIDS OCCURS. NOTE THE SYNCHRONY OF SEPARATION.

The spindle checkpoint controls chromatid timely separation SYNCHRONOUS SEPARATION OF SISTER CHROMATIDS IS THE CONSEQUENCE OF A SPECIFIC CONTROL MECHANISM EXERTED AT THE LEVEL OF KINETOCORES: THE SPINDLE CHECKPOINT. IT DETERMINES CHROMATID SEPARATION AT THE RIGHT TIME. In prophase kinetocore proteins are phosphorylated Bub1 e 3 (checkpoint protein) bind to kinetocores Mad2 binds to phosphorylated proteins of kinetocores Mad2 inhibits CDC20 WHEN CDC20 IS NO LONGER INHIBITED CDC20 activates APC/C APC/C degrades securin (separase inhibitor) Separase degrades cohesins Sister chromatids separate

A multivalency model for Mad1 kinetochore targeting. Kim S et al. PNAS 2012;109: ©2012 by National Academy of Sciences

APC (ANAPHASE PROMOTING COMPLEX) IS ACTIVATED BY CDC20 AND PROMOTES DEGRADATION OF SECURIN. THE ENZYME SEPARASE, NO LONGER INHIBITED BY SECURIN, DEGRADES COHESINS, WHICH HOLD CHROMATIDS TOGETHER. TRACTION FORCE EXERTED BY MICROTUBULES SEPARATES KINETOCORES. APC/C promotes chromatid separation by degrading securin

THE SHEME SHOWS AS LIKE APC ARE ACTIVATED BY CDC20 AND LIKE ACTIVATE APC DETERMINE DEGRADATION OF CICLINA M. APC IS AN UBIQUITINA LIGASE (E3) AND ACTS AFTER UBIQUITINAZIONE E1 ED E2 ENZYMES BONDING UBIQUITINA TO THE SUBSTRATE TO DEGRADE (LA CICLINA M, IN THIS CASE). DEGRADATION OF CICLINA, AND THE CONSEQUENT INACTIVATION OF M-CDK, ARE ESSENTIAL FOR TO PROCEED IN THE CELLULAR CYCLE. APC/C also promotes degradation of M-Cdk

19_18_envelope breaks.jpg Dephosphorylation of nuclear lamina proteins promotes the assembly of the nuclear envelope

IN PROPHASE-PROMETAPHASE NUCLEAR ENVELOPE DISORGANIZES. IN TELOPHASE NUCLEAR ENVELOPE RIORGANIZES. A CRITICAL ROLE IN BOTH PROCEDURES IS PERFORMED BY NUCLEAR LAMINA PROTEIN THAT SHALL BE PHOSPHORILATE IN PROPHASE BY M-CDK AND DEPHOSPHORILATE IN TELOPHASE BY A PHOSFATASES. PHOSPHORILATION PROMOTE THE DISORGANIZATION, THE DEPHOSPHORILATION AND THE RIORGANIZATION OF ENVELOPE MODULANDO THE INTERACTION BETWEEN NUCLEAR MEMBRANE AND CHROMOSOMES. NUCLEAR ENVELOPE DIS-ASSEMBLES AND RIASSEMBLES

IN ANIMAL CELLS CITOKINESIS (SEPARATION OF CYTOPLASM) IS ACHIEVED AS A RESULT OF THE CONTRACTILE RING OF ACTIN AND MYOSIN II THAT IS ORGANISED AT THE LEVEL OF THE EQUATOR OF THE MITOTIC SPINDLE. THE CYTOKINESIS IS EXACTLY REGULATED IN SPACE AND TIME. Cytokinesis in animal cels is promoted by a contractile ring

The contractile ring is made of actin microfilaments

RhoA regulates the assembly of the microfilament ring

ACTIN E MYOSIN II CONTRACTILE RING IS SHOWN IN FIGURE A, WHEN IS HIGHLIGHTED HIS ROLE TO DETERMINE A PROGRESSIVE CYTOPLASM THROTTLING. IN THE PHOTO B RING IS SHOWN AT ELECTRON MICROGRAPH, JUST BELOW THE PLASMATIC MEMBRANE. IN THE IMAGE C ACTIN AND MYOSIN ARE SHOWN IN RED AND GREEN THROUGH IMMUNOFLUORESCENT. THE CONTRACTILE RING CONTAINS ACTIN AND MYOSIN II

ACTIN E MYOSIN II RING PROGRESSIVELY CONTRACTS UP TO DETERMINE THE SEPARATION OF TWO DAUGHTER CELLS AS WILL BE EXPLAINED UP, CELLS IN COLTURE, OBSERVATED THROUGH SCANNING ELECTRON MICROOSCOPE. THE LOW IMAGE SHOWN LIKE APPEAR TO ELECTRON MICROSCOPE THE RESIDUAL BODY, THAT PORTION OF CYTOPLASM THAT STILL CONNECT TWO CELLS WHEN ACTIN RING IS CONTRACTED AND THAT, COMPLETED THE SEPARATION, CAN BEGIN TAKEN OVER BY ONE OF TWO DAUGHTER CELLS. CONTRACTILE RING

Cytokinesis is precisely regulated

THE EXPERIMENT DESCRIBED HERE SUGGESTS THAT THE SPINDLE POSITION IS CRITICAL FOR THE CORRECT POSITIONING OF THE ACTIN AND MYOSIN CONTRACTILE RING. The spindle determines the position of the actn ring

THE EXPERIMENT DESCRIBED HERE SHOWS THAT MYOSIN II IS ESSENTIAL FOR CYTOKINESIS. ALSO DEMONTRATES THAT MITHOSIS CAN INDIPENDENTLY UNWIND DALLA CYTOKINESI. IN FACT IF THE MYOSIN II FUNCTION IS INHIBITED BY DELETING THE GENE OR BY INJECTING AN ANTI-MYOSINII ANTIBODY, MITOSIS PROCEEDS WIHOUT CYTOKINESIS, LEADING TO A LARGE MULTINUCLEATE CELL. Myosin II is essential for the cytokinesis

The distribution of organelles among daughter cells HOW ARE ORGANELLES DISTRIBUTED AMONG DAUGHTER CELLS? In the secretory pathway the fusion processes stop while fission proceeds. Organelles of the secretory pathway fragment into vesicles that form clusters and are then segregated into daughter cells.

Evolution of the mechanism of chromosome separation

A HAPPY AND FRESH INTERPRETATION OF MITOSIS