BIO 121 Molecular Cell Biology Lecture Section 5

BIO 121 – Molecular Cell Biology Lecture Section 5 A. Review of the mitotic cell cycle and cell death B. Regulation of cell number and quality during mitosis C. Regulation of cell type during mitosis D. Wound Healing

Control of Cell Number A. Review of the mitotic cell cycle and cell death B. Regulation of cell number and quality during mitosis C. Regulation of cell type during mitosis

Numbers in a Cell Population • Cell number is a combination of. . • Cell divisions – Cell deaths (necrotic + programmed) • Necrosis is premature cell death – disease, injury, starvation, toxicity, excitotoxicity • Programmed cell death is death by design – apoptosis, anoikis, cornification, autophagy • Same for an organism, system, organ or tissue, and for single cell populations in an ecosystem

Programmed cell death is an essential component It guarantees appropriate ennervation patterns Figure 18 -13 Molecular Biology of the Cell (© Garland Science 2008)

Even gives us our fingers and toes! Figure Q 18 -1 Molecular Biology of the Cell (© Garland Science 2008)

Overview of the Mitotic Cell Cycle and Cell Division. Diploid cells duplicate the contents of their cytosol and nucleus prior to splitting to form two genetically exact daughter cells. Figure 17 -51 a Molecular Biology of the Cell (© Garland Science 2008)

We’ve learned to both control it. . A mutation in a signal molecule that limits muscle cell division has been bred in. Figure 17 -69 Molecular Biology of the Cell (© Garland Science 2008)

Fig. 11 -19 And fear it. . 2 µm A normal cell next to a tumor derived from uncontrolled cell divisions

Like everything else, the process of cell division has evolved over time

Fig. 12 -11 -1 Origin of replication E. coli cell Prokaryotic Division is called binary fission. Two copies of circular DNA, beginning at the origin of replication, actively move apart from each other. Two copies of origin Cell wall Plasma membrane Bacterial chromosome

Fig. 12 -11 -2 Origin of replication E. coli cell Two copies of origin Origin Cell wall Plasma membrane Bacterial chromosome Origin

Fig. 12 -11 -4 Origin of replication E. coli cell Two copies of origin Origin When the daughter chromosomes reach the opposite poles, the cell separates into two daughter cells Cell wall Plasma membrane Bacterial chromosome Origin

Fig. 12 -12 Since prokaryotes evolved before eukaryotes, mitosis probably evolved from binary fission. Certain protists exhibit types of cell division that seem intermediate between binary fission and mitosis. Bacterial chromosome (a) Bacteria Chromosomes Microtubules (b) Dinoflagellates Intact nuclear envelope Kinetochore microtubule Intact nuclear envelope (c) Diatoms and yeasts Kinetochore microtubule Fragments of nuclear envelope (d) Most eukaryotes

The complex mitotic cell cycle is used by the organism to control: cell number cell quality cell type

Target Mechanisms Cell Type Cell Quality Figure 15 -8 Molecular Biology of the Cell (© Garland Science 2008) Cell Number

We’ve even looked at one pathway already Figure 17 -62 (part 1 of 3) Molecular Biology of the Cell (© Garland Science 2008)

Fig. 12 -5 S (DNA synthesis) G 1 is ito MIT (M) OTIC PHA SE M C yt sis s e n i k o G 2

Interphase does the real work of mitosis • Gap 1 (G 1) is when the cell gets ready for DNA synthesis – Need DNA synthase complexes, repair enzymes, histones, etc. . • S-phase is when the cell synthesizes and edits chromosomes – Synthesis and error editing are the primary objectives. • Gap 2 (G 2) is when the cell gets ready for cell division – Need organelles, cytoskeletal proteins, molecular motors, metabolic enzymes, etc. – Kinetochores are refined and finalized.

Fig. 12 -UN 1 G 1 S Cytokinesis Mitosis G 2 MITOTIC (M) PHASE Prophase Telophase and Cytokinesis Prometaphase Anaphase Metaphase

M-Phase • Prophase: chromosomes condense with kinetichores, centrisomes move to poles, nuclear membrane disintegrates • Metaphase: spindle fibers attach and push to midline • Anaphase: kinetichores pull sister chromatids to poles • Telophase: the reversal of prophase activities • Cytokinesis: actin-based separation of cytosol into daughters

Sister chromatids are attached to each other at centromeres and each is individually attached to a kinetochore from the opposite pole metaphase Figure 17 -43 a Molecular Biology of the Cell (© Garland Science 2008)

Sister chromatids separate from each other, producing 92 individual chromosomes in humans, and are pulled by kinetichores to the poles. anaphase Figure 17 -43 b Molecular Biology of the Cell (© Garland Science 2008)

dynein motors Figure 17 -37 Molecular Biology of the Cell (© Garland Science 2008)

Figure 17 -40 Molecular Biology of the Cell (© Garland Science 2008)

Cytokinesis results from the assembly of an actin-myosin ring that gets smaller and smaller as mysoin pulls actin along actin. Ring assembly is microtubuledependent and myosin light chain kinase must be phosphorylated to begin. Figure 17 -49 a Molecular Biology of the Cell (© Garland Science 2008)

The cleavage furrow

Cell Division in Plants In some plants growth continues over the life of the organism

Regulation of the Cell Cycle • There are four major ‘checkpoints’ that monitor sensor systems and trigger molecular switches to get to next stage – – The G 1/S checkpoint – to enter the cycle or not The S-checkpoint – to synthesize DNA or not The G 2/M checkpoint – to divide the cell or not The M-checkpoint – to shift from metaphase to anaphase • The sensor systems are focused outside the cell as well as inside the cell to make sure conditions are appropriate • When ‘off’, the molecular switches halt progression, when ‘on’ they biochemically start the next stage

Fig. 12 -14 G 1 checkpoint Control system G 1 M M checkpoint G 2 checkpoint S G 2 The book claims 3 and describes 4, so we’ll go with 4

• For many cells, the G 1 checkpoint seems to be the most important one • If a cell receives a go-ahead signal at the G 1 checkpoint, it will usually complete the S, G 2, and M phases and divide • If the cell does not receive the go-ahead signal, it will exit the cycle, switching into a nondividing state called the G 0 phase Copyright © 2008 Pearson Education, Inc. , publishing as Pearson Benjamin Cummings

Fig. 12 -15 G 0 G 1 checkpoint G 1 (a) Cell receives a go-ahead signal G 1 (b) Cell does not receive a go-ahead signal

Stop and Go Signals at the G 1 Checkpoint • In multicellular organisms, the classic required external signals are growth factors, cytokines and hormones • Most cells also exhibit anchorage dependence, in which they must be attached in order to divide • Most cells also exhibit density-dependent inhibition, in which crowded cells stop dividing • Many single cells also monitor external nutrient availability and will not divide if it is inadequate Copyright © 2008 Pearson Education, Inc. , publishing as Pearson Benjamin Cummings

The Cell Cycle Switches at the First Three Checkpoints are Molecules Called Cyclins and Cyclin-Dependent Kinases Cyclins are expressed and degraded, when present the cell moves forward 1. G 1 and G 1/S cyclins regulate the G 1 checkpoint 2. S cyclins regulate S checkpoint 3. M cyclins regulate G 2/M checkpoint Cyclin-dependent kinases are always present and are activated by the binding of their appropriate cyclin Figure 17 -15 Molecular Biology of the Cell (© Garland Science 2008)

1. Cdk is always there, 2. if conditions are OK cyclin is expressed, 3. the combination is an active kinase, and 4. activity is lost with cyclin Cdk Cyclin accumulation S M Cyclin is degraded G 2 checkpoint MPF Cyclin

What does cyclin-cdk phosphorylate? • G 1 -cdk – Rb protein: loses inhibition of E 2 F – E 2 F then activates transcription of S-cyclin • M-cdk – histones: chromosome condensation – lamins: nuclear membrane fragmentation – myosin: purse-string cytokinesis

So, back to our growth factor example. . . What does immediate early gene expression cause? Figure 17 -62 (part 1 of 3) Molecular Biology of the Cell (© Garland Science 2008)

Immediate early gene, Myc, expression activates the expression of G 1 cyclin Figure 17 -62 (part 2 of 3) Molecular Biology of the Cell (© Garland Science 2008)

M Checkpoint Regulation • If any kinetochores are not attached to spindle microtubules during metaphase they send a molecular signal that delays cycle progression • The signal activates the anaphase-promoting complex, or cyclosome, known as APC/C • APC/C is a protease that destroys: – cyclins: stopping cell cycle – securin: which drives sister chromatid separation

Apoptosis During Mitosis Controls Cell Quality • Every cell cycle in multicellular organisms are molecularly hardwired with apoptosis as a potential outcome if things aren’t just right • The primary cause is imperfect DNA copying • This is one of our primary defenses against erroneous or uncontrolled proliferation

Characteristics: 1. Cessation of DNA repair mechanisms 2. Cell shrinkage 3. Nuclear membrane blebbing 4. DNA fragmentation 5. Death

Can result from bad copies during DNA replication Mdm 2 will cause p%# p 53 degradation Figure 17 -63 (part 1 of 2) Molecular Biology of the Cell (© Garland Science 2008)

p 21 has been shown to block cyclin/cdk activity at G 1 -S, S and G 2 -M Figure 17 -63 (part 2 of 2) Molecular Biology of the Cell (© Garland Science 2008)

The Roles of p 21 and PCNA • The apoptotic clock: – PCNA = the proliferating cell nuclear antigen – If [p 21] rises to 5 X [PCNA] apoptosis will happen – p 21 will reach binding affinity for PCNA – The dimer will shut off DNA repair mechanisms

When apoptosis is inactive, Bcl 2 is bound to APAF-1 and the dimer acts to block assemblage of mitochondrial membrane channel proteins, BH 123 (Bax and Bac) , Figure 18 -11 a Molecular Biology of the Cell (© Garland Science 2008)

The loss of DNA repair mechanisms: 1. Activates BH 3 -only proteins 2. Signal Bax translocation to the mitochondria

BH 3 -only inactivates Bcl 2/APAF-1 inhibition and along with Bax dimerization with Bac forms active channels Bax/Bac dimers apoptosis inducing factor Figure 18 -11 b Molecular Biology of the Cell (© Garland Science 2008)

Release of cytochrome C from the mitochondria outcompetes Bcl 2 for APAf-1 and activates assembly of the apoptosome Figure 18 -8 Molecular Biology of the Cell (© Garland Science 2008)

(8, 9, 10) (3, 6, 7) Figure 18 -5 b Molecular Biology of the Cell (© Garland Science 2008)

Characteristics: 1. Cessation of DNA repair mechanisms 2. Cell shrinkage 3. Nuclear membrane blebbing 4. DNA fragmentation 5. Death

Extrinsic apoptotic cascade also uses the common caspase cascade Figure 18 -6 Molecular Biology of the Cell (© Garland Science 2008)

3. Regulation of cell type during mitosis • Review – Symmetric vs. Asymmetric Cell Division – Stem Cell and Embryonic Cell Divisions • Regulation – Control in the Cytosol – Control in the Nucleus

Common Cell Divisions Produce Daughters Like the Parent to Replenish the Population Common during growth and the repair of damaged tissues

The Stem Cell Concept • Asymmetric division of stem cells produces one new stem cell and one differentiated daughter – Many stem cells in the embryo and adult • In some organs: frequent replenishing divisions – gut, epidermis, bone marrow – example: billions of blood cells are destroyed by the spleen every hour • In others, they only divide in response to stress or the need to repair the organ – heart, prostate

Stem Cell Mitosis HSC Each division produces daughter cell(s) unlike the parent cell: - The first two are asymmetric and produce one stem cell - The last one produces two like daughters unlike parent

Stem Cell Types: Embryonic Stem Cells The Inner Cell Mass Produces all cells of the embryo

Stem Cell Types: Adult Stem Cells • Committed stem cells with limited potential – hematopietic stem cells - hair stem cells – mesenchymal stem cells - melanocyte stem cells – epidermal stem cells - muscle stem cells – neural stem cells - tooth stem cells – gut stem cells - germline stem cells – mammary stem cells

The hair shaft is composed of keratinocytes, lubricated by sebaceous secretions, with melanin for color. - “Bulge” is the stem cell niche for hair basal cells, sebocytes and melanocytes. - The first two arise from a common stem cell but melanocytes arise from a committed stem cell.

Hematopoietic stem cells in the bone marrow are the kings of differentiation choices Progenitor cells will produce two daughters like each other but not like the parent, with the choice being determined by the current, local signaling combination

Control of Assymetrical Division in the Cytosol • G 2 is the key to differentiation events • The daughter cells can be built differently – DNA is the same but. . – RNA’s can be different – Proteins can be different – Cytoskeleton and organelles can even be different • The parent cell must focus its placement of these components on either side of the furrow

Isolation of transcription factors across midline

Fig. 18 -15 a Unfertilized egg cell The unfertilized egg is the queen Sperm of cytoplasmic isolation of cell Fertilization fate determinants Nucleus Two different cytoplasmic determinants Zygote Mitotic cell division Two-celled embryos have two different cell types All stem cells do it effectively

We use the embryonic process of cleavage to disperse the careful distribution of cytoplasmic determinants laid down in the egg.

The G-phases of somatic mitosis allow for cytoplasmic growth so that the daughter cells are equal in size to the parent cell. In cleavage we want to use the egg cytoplasmic material so we just skip the G-phases all together. It makes the cell cycle go very fast! - Frogs can make 37, 000 cells in 43 hours. - Fruit flies can make 50, 000 in 12 hours (10 min!)

They don’t even bother to make plasmamembranes until later!

Control of asymmetric division in the nucleus

Two DNA methyltransferases are important in modifying DNA

Development of pluripotency in the inner cell mass depends on methylation pattern Both parent gametes have methylation patterns that must be removed for all genes to be available to the developing organism.

One of the big hurdles to somatic cell nuclear transfer cloning was overcoming the adult methylation pattern It took hundreds of failed attempts before the successful cloning of Dolly from adult mammary epithelium.

Each step of differentiation of a given cell type depends on changing methylation patterns These genes maximize effectiveness when coding for transcription factors, SNu. RPs, signaling receptors, etc.

Cutaneous Wound Healing The skin is a complex organ. . .

Many cells and activities involved

Many cells and activities involved in Healing Clotting Scarring Re-establishing Function

• Four overlapping stages to wound healing – Hemostasis – Inflammation – Proliferation – Maturation

Blood flows into the exposed ECM of the injured tissue.

RBC and Platelets Trapped in Fibrin Clot

Clotting factor VII from the blood contacts tissue factor on cells in the damaged tissues to activate clotting

. Platelet activation in the clot makes them sticky and releases their signal storage

Positive feedback activates even more

Platelet activation releases growth factors by regulated secretion

Inflammation is a process mediated primarily by WBC as part of our innate immunity - Resident mast cells and macrophages - Recruited monocytes and neutrophils

Resident mast cells also degranulate rubor = redness calor = heat tumor = swelling dolor = pain

Activated mast cell activities

Figure 1 Development and differentiation of macrophages. Rickard A J , Young M J

Activated macrophage activities

The special case of extravasation • Circulating WBC must get out of the vessel • Combines activation of the WBC with ‘Cell Rolling’, ‘Adhesion’ and ‘Diapedesis’ 1. The presence of environmental cues associated with injury and infection change endothelial surface selectins 2. These catch closely matched WBC surface oligosaccharides and make them roll to a stop on endothelial surface 3. The white blood cell then activates an integrin that binds tightly to ICAM on endothelial cells 4. Diapedesis uses basic migratory mechanisms along with WBC shape change to squeeze between endothelium

Intercellular Diapedesis

Transcellular Diapedesis

Activated neutrophils are phagocytic

Pseudopod-Driven Phagocytosis in Neutrophils Figure 13 -46 Molecular Biology of the Cell (© Garland

Actin polymerization is required to extend pseudopods Actin depolymerization is required to seal off the phagosome Figure 13 -47 a Molecular Biology of the Cell (© Garland Science 2008)

Proliferation re-establishes tissue function • Reconnection of the dermal connective tissue • Integrity of the epidermal layers • Re-establishment of blood flow

Reconnection of the dermal CT

Cell Migration or “Crawling” • The Basic Mechanism – Triggered by signals from outside the cell – Actin-myosin based movement – Requires attachments to outside to pull against – Gotta’ drag all of the cell contents along for the ride

Chemotaxis Circumferential receptors Rho-family GTPases (monomeric) Rho-dependent kinases 1. Actin monomer nucleotide exchange 2. Actin fiber polymerization and disassembly 3. Myosin motor ATPase activity