1. Lecture 22: Mechanisms of cell division “Omni cellulae e cellulae…” (all cells come from cells) Virchow circa 1855 Prokaryotes Eukaryotes = M phase mitosis - separation of chromosomes cytokinesis - splitting of the cytoplasm

2. Prokaryotes divide by septation and fission Localized insertion of new membrane and cell wall components Septum Single chromosome attached to membrane… Replicate DNA… Daughter chromosomes attached to membrane… Localized wall and membrane growth segregates daughter chromosomes… Septum Mitochondria and plastids divide by fission!

3. Eukaryotic cell grow and replicate DNA and organelles during “Interphase” (= G 1 + S + G 2 ) Appearance: Nuclear envelope intact and chromatin dispersed (decondensed) Interphase MT array Key functions: Most cell growth and organelle biogenesis occurs during interphase DNA replicates (in S) Centrosomes duplicate ECB Panel 19-1 Intact nuclear envelope Centrosome and radial MT array Dispersed chromatin

4. G1G1 G1G1 MG2G2 S Animal cells inherit a single centrosome (centriole pair) from previous division Centrosome duplication is initiated late in G 1 by G1-S Cdk trigger Parent centrioles separate (disjoin) and centrioles replicate in a template-dependent process during S phase Daughter centrosomes function as spindle poles during M-phase Animal cells must duplicate their centrosomes prior to division Parent centrosome contains a pair of centrioles Disjunction Daughter centrioles Centriole replication

5. 2. “Prometaphase” (before the middle) Nuclear envelope disassembles Chromosomes attach to spindle 3. “Metaphase” (the middle) Chromosomes align at metaphase plate 5. “Telophase” (the end) Nuclear envelope reassembles Cytokinesis begins 1. “Prophase” (the beginning) Chromosomes condense Spindle assembly begins Overview of “mitosis” and “cytokinesis” in eukaryotes Mitosis (nuclear division) consists of five phases: Cytokinesis (cytoplasmic division) Actin and myosin II in animal cells MT phragmoplast in plant cells Division must partition all organelles (ribosomes, ER, Golgi, lysosomes, mitochondria, centrosomes, etc) and components to “daughters”… Organelles with high copy number (mito, chloro, ribosomes, ER, Golgi) segregate by mass action Organelles with low copy number (chromosomes, centrosomes) use specific segregation mechanisms 4. “Anaphase” (after the middle) Chromosomes segregate 19.2-animal_cell_division.mov 19.1-plant_cell_division.mov

6. Gr = MTs Bl = DNA Mitosis begins with “prophase” Centrosome separation beginning Nuclear envelope intact Condensing chromosomes attached at centromeres Duplicated centrosomes separating to act as spindle poles; driven by MT motors (kinesins) Intact nuclear envelope Centrosomes Condensing chromosomes Intact nuclear envelope ECB Panel 19-1

7. Chromosomes condense during prophase Duplicated chromosomes consist of two “sister” chromatids Each contains a single DNA molecule, 2 DNA molecules are identical Sister chromatids are linked at centromere by cohesin proteins (earlier, in S, cohesion along entire chromatid length) Early prophase Metaphase chromosome ECB 19-3 Centromere 2 “sister chromatids” Cohesins

8. “Prometaphase” begins with nuclear envelope breakdown M phase Cdk (MPF)-dependent phosphorylation of nuclear lamins induces disassembly of nuclear lamina and envelope breakdown Chromosomes are “dumped” between poles of spindle Chromosomes attach to “spindle” by “capturing”spindle MTs Remnants of nuclear envelope Chromosomes capture spindle MTs ECB Panel 19-1

9. Spindle MTs are captured by kinetochores Sister chromatids are linked at centromere by cohesin proteins Each chromatid assembles a kinetochore at the centromere Kinetochores capture + ends of MTs (1-40) to attach chromatids to opposite spindle poles Spindle MTs are 20X more dynamic than interphase MTs (stabilizing MAPs phosphorylated and removed: catastrophins) Metaphase chromosome ECB19-3 Centromere 2 “sister chromatids Cohesins Kinetochore Inner plate Outer plate Kinetochore MTs (1-40 KT MTS make “KT fiber”) ++ ECB 19-9 MTs attached to kinetochore

10. “Prometaphase” chromosomes are under tension ECB panel 19-1 Spindle poles Ch romosomes may initially capture MTs from a single pole Eventually, chromosomes attach to MTs from both poles (sister chromatids to opposite poles) In “congression,” chromosomes oscillate between spindle poles Pulled and/or pushed? MT assembly and disassembly or motors? Both kinesins and dynein localized to “kinetochore”…

11. “Metaphase” Metaphase plate Chromosomes align at the “metaphase plate”: a plane ~equidistant from each spindle pole. Alignment results from a balancing of forces from each spindle pole MCB figure 19-35C Metaphase plate Pole Metaphase plate ECB Panel 19-1

12. Metaphase spindles in animal cells include 3 classes of MTs Overlapping interpolar MTs Spindle pole (centrosome) Astral MTs Kinetochore fiber “Kinetichore fibers” (= 1-40 kinetochore MTs) link chromosomes to the poles… Interpolar MTs extend from the spindle poles and interdigitate in central spindle; interacting MTs become stabilized Dynamic “Astral MTs” extend radially from spindle poles… Kinetochore Sister chromatids Note that all MTs have plus-ends away from centrosome/spindle pole + + + + + + + + + + + + + + + + + + + + + + + + + + + + + See ECB 19-7 and 19-13 19.4-mitotic_spindle.mov

13. MCB figure 19-35D Chromosome segregation occurs during anaphase Metaphase plate Pole Metaphase plate Anaphase A: Chromatids separate and move towards poles at ~1  m/min -1 Anaphase B: Poles separate ECB Panel 19-1

14. Sister chromatids separate in anaphase Anaphase A and B may temporally overlap Anaphase promoting complex (APC, last lecture) triggers degradation of cohesin proteins linking sister chromatids at centromere Chromatids jump apart and then move more steadily Astral MTs Kinetichore fiber Sister chromatids Sister chromatids move towards opposite spindle poles in Anaphase A and spindle poles separate in anaphase B ECB 19-17

15. Multiple forces may contribute to chromosome movements in Anaphase A Overlapping polar MTs Motors Spindle pole (centrosome) Astral MTs Kinetichore fiber 1. Shortening (disassembly) of MT at kinetochore may provide poleward force 2. Minus-end directed motor (dynein?) at kinetochore may provide poleward force ECB 19-17 Both?

16. Multiple forces may contribute to pole separation during Anaphase B Overlapping polar MTs Motors Astral MTs 1. Pulling forces generated by astral MTs interacting with (attaching to) cell cortex 2. Motor-dependent sliding of anti-parallel polar MTs in spindle mid-zone (coupled with elongation of mid-zone MTs) Which motor do you guess? 3. Both #1 and #2

17. A “spindle checkpoint” monitors chromosome attachment APC Extra chromosome Missing chromosome Metaphase Cytokinesis Anaphase XX Unattached kinetochore Normal chromosome segregation With checkpointIf there were no checkpoint Delay provides time for normal attachment Unattached kinetochore inhibits APC

18. Lamins are dephosphorylated Nuclear envelope reformation begins around individual chromosomes which then fuse to form daughter nuclei Interphase MT array reforms Contractile ring at division furrow… Cytokinesis in animal cells Nuclear envelope reforming around individual chromosomes… Contractile ring of actin/myosin forming… Daughter chromosomes reach separated poles Nuclear position determines division plane but mechanism unclear Actin filaments (red) Myosin filaments (green) ECB Panel 19-1 Cytokinesis begins as actomyosin contractile ring divides cytoplasm Sliding of actin and myosin creates “purse-string” contraction

19. Summary of mitosis and cytokinesis in animal cells 1. Prophase Nuclear envelope intact… Chromosomes condense… Centrosomes separate… MTs VERY dynamic… 2. Prometaphase NE disassembles… Kinetichores capture MTs… Chromosomes attach to spindle… Congression movements… Interphase (G 1 -S-G 2 ) Cell growth.. Organelle biogenesis… DNA replication (S-phase)… Centrosome duplication… MTs dynamic… 3. Metaphase Chromosomes align at metaphase plate… 4. Anaphase Chromatids segregate… A: chromatids to poles… B: poles separate… 5. Telophase NE re-assembles… Chromosomes decondense… Cytokinesis begins… Cytokinesis Contractile ring of actin and myosin divides cell… Daughter cells enter G 1 …

20. Mitosis and cytokinesis in higher plants During interphase, MTs are organized in circumferential bands These guide cellulose deposition (next slide) Spindle poles are less focused and spindles are barrel-shaped Higher plants lack centrioles and conventional centrosomes Centrioles lost when cell motility lost? Before mitosis, a “pre-prophase band” of MTs marks the future division site How it forms is unclear And it disappears before mitosis! Cell wall Pre- prophase band of MTs  -TB, etc ECB 19-22

21. Cell wall dictates direction of cell elongation in interphase plant cells Cellulose in wall Cell only grow (elongate) perpedicular to cellulose Microtubule in cytoplasm Microtubules in cytoplasm are colinear with cellulose in wall Thought that MTs somehow control orientation of cellulose deposition ECB 21-6

22. Microtubules may guide cellulose synthase movement in plane of membrane

23. Rigid cell wall requires a distinctive mode of cytokinesis in plants Division is completed with fusion of the cell plate with the plasma membrane and cell wall Circumferential MT array is re- assembled Cell plate (membrane and nascent cell wall) phragmoplast Vesicles fuse to ends of growing cell plate During late mitosis, golgi vesicles containing cell wall precursors are transported towards the division site Vesicle fusion initiates assembly of the “cell plate” (the division membrane and nascent wall) As vesicles fuse, the cell plate grows outward towards the cortex The “phragmoplast,” a ring of MTs surrounding the cell plate guides deposition of cell plate ECB 19-22

24. A comparison of mitosis and cytokinesis in animals and plants Centrosomes act as spindle polesHigher plant cells lack “classical” centrosomes… “Anastral” spindle is barrel-shaped Contractile ring of actin and myosin… Plant cells surrounded by rigid wall MT “phragmoplast” organizes growing cell plate PlantsAnimals Cytokinesis Mitosis phragmoplast

25. A comparison of chromosome segregation in diverse taxa Adapted from MBoC figure 18-40 There are many variations on the theme!