Nucleus/Cell Division

anucleated cells examples

mammalian RBCs, skin cells, lens fiber cells

nuclear characterization of mammalian, avian, amphibian RBCs

mammals: anucleated

avains and amphibians: nucleated

lens fiber cells

differentiate from lens epithelial cells at equator (germination layer)

-functions throughout life --> could lead to blockage

benefit of lens fiber cells being anucleated

no nucleus to diffract light as it passes through lens

nuclear envelope

layer of two membranes that surrounds the nucleus of a cell

-50% nuclear pores

nuclear pores

holes in the nuclear envelope that allow materials < 62,500 Da to pass in and out of the nucleus

proteins unable to pass through nuclear pores

require ATP/receptors of their own

nucleoplasmin

pentramer chaperone protein involved in chromatin assembly and genetic stability

-10% of total protein in X. laevis

first molecular protein discovered

nucleoplasmin

X. laevis

African clawed frog

-10% of total protein makeup is nucleoplasmin

lamins

intermediate filaments surrounding the nuclear envelope that are involved in shaping the nucleus

-A,B,C

lamins and cell cycle

involved in dissolution of nuclear envelope in mitosis

-laminin B phosphorylated by MPF

lamin B

phosphorylated by MPF during prophase to dissolve nuclear membrane

progeria

rapid aging diseased cause by defects in lamin A (LMNA gene)

farnesyl

molecule added to lamin A protein to build nuclear lamina

-cut off to allow lamin A to be incorporated into lamina

progeria nucleus

farnesyl molecule attaches to lamin B but does not detach --> piles up at nuclear envelope

lonafarnib

farnesyltransferase inhibitor used to treat progeria

embryogenesis

the process by which a single-celled zygote becomes a multicellular embryo

-humans go from zygote to ~100 trillion cells

cross-talk of cells and cell division/death

cells cross-talk to establish balance between cell division and cell death

-skin cells, RBCs

G0 phase

a nondividing state occupied by cells that have left the cell cycle, sometimes reversibly

G0 phase triggers

lack of mitogens

-antiproliferation signals, contact inhibition, TGF-beta telomere damage

G1 phase

stage of interphase in which cell grows and performs its normal functions

Arthur Parfee

growth factors required to get through G1 in specific order and timing

-3T3 cells

3T3 cells

mouse fibroblasts

-used by Arthur Parfee to understand G1 phase

growth factors essential to G1 phase

in order: PDGF, EGF, insulin

early- and late- response genes in G1 phase

late response genes rise in expression after peak of early response genes

-late-response genes dependent of degradation of early response

early- and late-response genes in G1 phase experiment

blocking degradation of early-response proteins: early response expression remains constant, late response proteins never rise

-late-response genes dependent on activity of early-response genes

cell synchrony

collecting cells undergoing cell stages at the same time to study

importance of cell synchrony

G1 phase is most variable in time, so cells don't always stay in perfect synchrony

cell synchrony techniques

G1 phase: remove cell culture serum or amino acids (growth factor deprivation) or add reversible protein synthesis inhibitors

M phase: reverse microtubules inhibitors

S phase: DNA synthesis inhibitor

restriction point (Pardee point)

the point in the G1 stage where the cell is committed to continue through the rest of the cell cycle and divide

checkpoint

a control point in the cell cycle where stop and go-ahead signals can regulate the cycle

-checks fidelity of cell division

cyclins

proteins that control cell cycle transit

Ruderman and Hunt

discovered cyclins

-used sea urchin embryos

Ruderman and Hunt and sea urchins

sea urchin embryos --> fertilize --> SDS gel of cells at certain points to see the change in protein levels

-several proteins (cyclins) found to rise and fall during cell division

cyclins and yeast

yeast have similar types of cyclins in cell division than those of humans

cyclins A and B

mitotic cyclins; most abundant during mitosis

mitotic cyclins

interact with cyclin-dependent kinase to phosphorylate cell division-associated proteins

-cyclins A and B

cyclin E

G1/S phase

cyclin D

required for G1 transit

cyclin D experiment with blocking antibody

add growth factor to G0 cells -->

-control: add BrdU --> BrdU-positive cells after 16 hrs

-treatment: microinject anti-cyclin-D antibody at different times --> higher concentration of BrdU-negative cells among cells that were injected earlier

BrdU

stains the DNA of dividing "newborn" cells

-alternative to 3H-thymidine

S phase

DNA synthesis and replication

-helicase, DNA synthase; bidirectional

replicon

unit of replication, consisting of DNA from the origin of replication to the point at which replication on either side of the origin ends.

proving bidirectional nature of S phase

tag cells with BrdU or 3H-thymidine --> observe during S phase --> 3H tag or BruD expanding in opposite directions from the replicon

G2 phase

verifies that all DNA has been correctly duplicated and that DNA errors have been corrected

-chromosome condensation, early organization of cell cytoskeleton

mitotic cyclin dependent kinases initiate

G2 phase

M phase

mitosis and cytokinesis

-shortest yet most dramatic phase

-reassembly of nuclear envelope; MPF

reassembly of nuclear envelope

new membrane comes from ER

maturation promoting factor (MPF)

mitotic cyclin-CDK complex responsible for phosphorylating lamin B during M phase

-dissolution of nuclear envelope

MPF cycle

tagged with ubiquitin in late anaphase --> degradation --> synthesis of mitotic cyclin once levels become relatively low --> phosphorylate lamin B -->

MPF frog experiment

inject cytoplasm from frog egg arrested in metaphase of meiosis II and MPF into oocyte arrested in G2 --> premature M phase

mitosis phase factor

mitotic cell (MPF) and G1-phase cell fuse --> premature M phase

cell cycle stages times (24-hr cycle)

G1: 9 hrs

G2: 4 hrs

M: 30 mins

measuring G1 transit time

synchronize cells

-add 3H-thymidine at start of G1 --> wait for cells that show 3H in S phase --> record duration

measuring S phase transit time

randomly synchronized cells

-note ratio of 3H-thymidine cells to normal cells and multiply by 24 (hrs)

measuring G2 phase transit time

random cycling

-add 3H-thymidine to S-phase cells --> wait for 3-H M cells to arise

measuring M phase transition time

identify easily in cell culture of randomly-cycling cells

-note ratio of M phase cells to other cells and multiply by 24 (hrs)

protein synthesis involved in cyclic behavior of cell division experiment

beaker: cytoplasm from activated egg + ATP + nuclei --> cells divide in time

-control: mitotic CDK activity/cyclin concentration rise during early mitotic events, decrease during late mitotic events

-add RNase: Mitotic CDK activity/cyclin concentration not present

-RNase-treated extract + wild type mitotic cyclin mRNA: Mitotic CDK activity/cyclin concentration increases, decreases cyclically

Ruth Sager

there are genes that suppress the development of cancer cells (tumor suppressor genes)

Ruth Sager experiment

fused normal and cancerous cells --> produced cell with normal phenotype --> many generations pass --> cell with cancer phenotype

p53

tumor suppressor gene causing cell cycle arrest in G1

-provides time for DNA repair (apoptosis if too far gone)

p53 pathway

DNA damage --> ATM/R phosphorylated p53 --> p21 activation --> stops G1 CDKs

ATM/R

phosphorylates p53 following DNA damage

guardian of the genome

p53

budding and fission yeast

used to understand cell cycle

-divide unevenly-budding or evenly-fission

cyclins are dependent on

cyclin-dependent kinases