CELL IV(30% of Block 2 Test)

Cell Locomotion: Reasons for locomotion of cells

Embryonic development. (Organogenesis and morphogenesis)

Physiological/Pathological Leucocyte margination Platelet adhesion Wound healing Metastasis (Cancer)

Physiological/Pathological Leukocyte margination:

Adhesion to endothelial cells Platelet Adhesion

Platelets adhere, degranulate, initiate clotting Wound Healing

Integrins cell adhesion cell movement and migration.

enable epithelial cells to migrate during wound closure aid signal transduction

epidermal cells migrate into the wound

changing the number, type, and distribution of integrins in the cells Metastasis:

Proliferation of the primary tumor; intravasation

invasion of the blood or lymphatic circulation Structures involved for movement

Lamellipodium by Rac (Rho family of GTP-Binding proteins) undulating motion,

ruffled appearance. Seen in fibroblasts

Filopodia-activated by Cdc42 (Rho family of GTP-Binding proteins) actin filaments. extension & retraction movements. Seen in growth cones of axons.

Cell Locomotion involves three processes: Lamellipodia

extend from front of the cell. Attachment:

Actin cytoskeleton makes contact with substrate. Traction:

Body of the cell moves forward. Cell Cycle:

Cell Division: Key Roles:

Genome: cell’s genetic information Somatic (body cells) cells Gametes (reproductive cells): sperm and egg cells Chromosomes: DNA molecules Diploid (2n): 2 sets of chromosomes Haploid (1n): 1 set of chromosomes Chromatin: DNA-protein complex Chromatids: replicated strands of a chromosome Centromere: narrowing “waist” of sister chromatids Telomere: ends of the chromosomes Mitosis: nuclear division Cytokinesis: cytoplasm division Meiosis: gamete cell division

Number of Chromosomes: Haploid (23) – ‘n’ number of chromosomes Diploid (46) – ‘2n’ number of chromosomes

Amount of DNA with respect to cell division: Haploid – ‘N’ amount of DNA Diploid – ‘2N’ amount of DNA

Interphase: All somatic cells: 2n,2N.

(n = the number of sets of chromosomes, N = the amount of DNA)

Interphase (2n,2N→2n,4N): Phase during which cells do not undergo division .

Mitosis: Prophase

chromatin condenses into chromosomes (visible) Nucleolus and the nuclear envelope begin to disappear Kinetochores will develop as microtubule organizing centers Prometaphase

Begins when the nuclear envelope disappears Chromosomes are randomly dispersed in the cytoplasm Chromosome microtubules attach to kinetochores

Kinetochores attach to spindle microtubules Kinetochore microtubules

Metaphase Chromosomes align at equator, homologs align independently of each other

Anaphase Anaphase promoting complex

APC inhibits securin Securin is involved in cohesion of sister chromatids APC is a target for anti-cancer therapy

Spindle elongates Shortening of microtubules—movement of chromosomes Chromosomes are split in synchrony Chromatids separate at the centromere Daughter chromosomes move to opposite poles of the cell Cleavage furrow begins to form

Contractile ring-- actin filaments Telophase

Each set of chromosomes reaches the pole Deepening of the cleavage furrow Cytokinesis Nuclear envelope is reestablished Nucleoli reappear Daughter cells enlarge Chromosomes disperse to form the typical interphase nucleus

Meiosis Prophase (subdivided into 5 stages)

Prophase I: Leptotene Chromosomes appear as threads (diploid number).

homologous chromosomes are unpaired Prophase I: Zygotene

Homologous chromosomes paternal and maternal arrange lengthwise (pre-synapsis)

Prophase I: Pachytene Chromosomes become shorter and thicker in appearance most easily identifiable stage of primary spermatocytes. Chromosomes are completely paired Synaptonaemal complex (SC): synapsis

Prophase I: Diplotene Dissolution of synaptonemal complex Chromosomes are shorter Homologous chromosomes repelling one another “Crossing Over” of chromatids arms -- Chiasmata chromosomal separation

chromosomal abnormalities may occur at this time Prophase I: Diakinesis

Continued separation of chromosomes nuclear envelope and nucleolus disappears.

Metaphase I

Anaphase I Telophase I 2nd Meiotic Division

Prophase II – Interkinesis without duplication of the chromosomes

Metaphase II Anaphase II Telophase II

Cell Cycle G1-phase (2N DNA, 46 chr)

Continue dividing variations in length of G1 phase

Or exit dividing. Enter GO phase (2N DNA, 46 chr)

Neurons are usually in G0 phase S-phase (2N / 4N, 46 chr)

DNA replication G2-phase (4N (tetraploid))

reached following completion of DNA replication. M-phase (4N)

mitotic phase Flow Cytometry

Thymidine incorporation 3HT=>autoradiograph technique

cell cycle based on the intensity of fluorescence of the nuclei DNA content of 2N

Most Cells large peak on the flow histogram.

DNA content of 4N 3.4% of the cell cycle

peak is much smaller Cell Cycle Checkpoint Genes (Rare genetic mistakes)

If a genetic mistake (i.e. exposure of a cell to radiation ) cell cycle checkpoint genes

prevent cells from dividing into two daughter cells. G1 Checkpoint

Adequate cell size Sufficient nutrients Growth factors present

G2 Checkpoint Adequate cell size Successful chromosomal replication

Metaphase Checkpoint All chromosomes attached to mitotic spindle

Cell Cycle Regulators Cyclins (rise and fall with the stages of the cell cycle)

G1 phase cyclin D S-phase cyclins E and A

mitotic phase cyclin B Cyclin-dependent kinases (Cdks)

levels in the cell remain fairly stable bind the appropriate cyclin for activation

G1 phase Cdk4 S-phase Cdk2 M-phase Cdk (Cdk1)

Phospholates protein substrates control processes in the cell cycle

Anaphase-promoting complex (APC). also called the cyclosome or APC/C triggers destruction of the cohesins

degrades the mitotic (B) cyclins sister chromatids to separate

Growth Factors: Involved in G1 checkpoint

Retinoblastoma protein (Rb) Tumor suppressor protein Bound to E2F (Transcription factor)

Shuts down cell cycle Growth Factors activates cyclin/cdk complex

Phospholates Rb E2F is released

E2F stimulates S phase protein production Cell Cycle Checkpoint Regulation:

p53 – pro-apoptotic tumor-suppressor gene, Protein p53

Minor DNA damage halts the cell cycle until repaired.

Major DNA damage cannot be repaired triggers apoptosis.

Protection against cancer - acts as a tetramer Single mutation can result in a dominant negative >half of human cancers

p53 mutations no functioning p53 protein.

G1 response to DNA damage. DNA damage blocks entrance into S phase.

Phosphorylation of active ATM activates pathways to G1 arrest

Chk2 phosphorylates Cdc25A inactivation and inability to activate CDK2

Phosphorylation of p53 protein transcription of a number of genes

DNA repair and cell survival. Phosphorylation and inactivation of Mdm2

Stabilization of p53. p16INK4a (product of the tumor suppressor gene INK4a

Inhibits cyclin-dependent kinase Cdk4. Increasing amounts of p16INK4a with age

Reduces the risk of uncontrolled mitosis, i.e., becoming a cancer. Deletions and other mutations of p16INK4a are found in a variety of

cancers However, ability to reproduce and thus replace lost or damaged tissue

diminishes. Mutations Causing Loss of Growth-Inhibiting and Cell-Cycle Controls

Normally a balance between growth-stimulating and growth- inhibiting signaling pathways.

Overproduction of cyclin D, a positive regulator, or loss of the negative regulators p16 or Rb, commonly occurs in human cancers.

Micro RNAs (mi RNA ) Regulate oncogene expression Transcribed into RNA (but not protein) mRNA complementary Noncoding intron regions of DNA Expression of miRNA altered in tumors

over expression of oncogenes Molecular Biology of Cancer

Proto-oncogene Non- mutated normal cellular genes

Oncogene Mutated genes responsible for cancer development

usually become dominant Examples

Translocation Chromosome fragments rejoin incorrectly

Philadelphia chromosome (Bcr-Abl gene) give rise to chronic myelogenous leukemia (CML) Gleevec / imatinib mesylate

Amplification Increased copies of proto-oncogenes

high expression of the normal protein Proto-oncogene point mutation

protein product more active more resistant to degradation

Tumor suppressor genes, (typically recessive) Negative regulators of the cell cycle (Rb, p16). Receptors or signal transducers that inhibit proliferation (TGFβ). Checkpoint mutations (p53). Proteins involved in DNA repair XP, FA, BRCA) proteins that reverse or prevent DNA damage (caretaker genes)

As multiple genetic abnormalities occur, multiple sub-clones develop or evolve. Eventually, one of these sub-clones may acquire the necessary combination of genetic abnormalities to become a cancer

Chromosomal errors, I Nondisjunction :

Chromosomes do not separate properly during meiosis I or sister chromatids fail to separate during meiosis II

Maternal age effect Older women have higher incidences Eggs being held in a meiotic block (meiosis II) for an extended period of time

Paternal age effect Older men can produce sperm with abnormal chromosome numbers

Aneuploidy: chromosome number is abnormal Monosomy~ missing chromosome

Turner Syndrome Lacks X or Y sex chromosome ~45 XO Exhibit female phenotype; sterile Short stature, webbed neck; high arched palate Cognitive defects (i.e., affects learning) Occurrence: 1 in 2500 females

Most cases of monosomy are not viable Trisomy ~ extra chromosome

Down syndrome-trisomy 21 Extra copy of chromosome 21 ~ 47,XX +21 or 47, XY +21

Klinefelter Syndrome: 47, XXY Male with extra X chromosome

Polyploidy~ extra sets of chromosomes 69 XXX, XXY, or XYY

Chromosomal errors II Deletion: removal of a chromosomal segment Duplication: repeats a chromosomal segment Inversion: segment reversal in a chromosome Translocation: movement of a chromosomal segment to another

Genomic Imprinting Parental effect on gene expression Identical alleles have different effects on offspring

zygote via the ovum or via the sperm. Fragile X syndrome

high prevalence retardation in males Mechanisms of cellular homeostasis / cell death

Proteosomes Cellular homeostasis, and in human disease Enzymes of the ubiquitin (non-lysosomal) protein degradation

ATP-dependent Degradation of all regulatory proteins

regulatory proteins are key to the activation or repression of -cycle progression, transcription, and apoptosis.

Proteasome inhibitors are currently under investigation as cancer therapeutic agents

Degrade misfolded proteins May contribute to neurodegenerative diseases

Employed in control of the cell division cycle and cell growth

Produce peptides six to nine amino acids in length major histocompatibility complex I peptides to

induces antibodies. Calcium and intracellular signaling

Low concentration maintained Calcium transport pump

Endoplasmic reticulum (ER)=>Cytosol=>plasma membrane Calcium-binding proteins

Activated by high Ca++ concentrations Calmodulin (CALcium MODULated proteIN) (CaM)

Ca++ concentration increases from 0.1 to 0.5 micro molar Binds target proteins

Many not able to bind to Ca++ directly Protein phosphatases

Removal of phosphate group Protein kinase

Attaches phosphate group Ca2+/calmodulin-dependent protein kinases (CaM Kinase)

Involved in many signaling pathways Cyclic nucleotide metabolism (cAMP) Transduction pathways involving phosphorylation Calcium transport (plasma membrane Ca2+ pump) Nitric oxide pathway Regulation of cytoskeletal proteins Apoptosis

Calcium-dependent cysteine proteases Calpain protease

Activates cathepsin Necrosis (Unprogrammed cell death)

Apoptosis (programmed cell death) Elimination of cells no longer necessary (larval tissue) or unwanted (tissue

between the digits) Brain development to remove neurons Defense mechanism

virus-infected cells damaged cells.

balancing cell proliferation Mediated by signaling pathways Major steps involved

Chromatin, nucleus, and cell condense Nucleus is broken apart Blebbing of plasma membrane Fragmentaion into apoptotic bodies Rapid phagocytosis

Mitochondria and lysosomes involvement mitochondrial membrane permeabilized

cytochrome c leaks into the cytosol Lysosomes leak proapoptotic proteins

Cysteine cathepsin

Activation of caspase protease => cell death Caspase (Intrinsic pathway)

Regulated by the Bcl-2 family Ultimate effectors of apoptosis Cysteine residues at their active site

Cleave Aspartic Acid substrate protein. Cell death Receptors (extrinsic pathway)

Receptors that directly induce cell death secrete polypeptides to signal apoptosis

Tumor necrosis factor (TNF) Fas receptor TNF- also mediates other signaling pathways involved in normal α

cellular functions. Cell Differentiation

Derived from a single fertilized ovum Fetal development, cell differentiation

Morphological The way the cell looks (e.g., RBC is round)

Biochemical Differentiation New biochemical components such as proteins (e.g., Hemoglobin in RBC)

Molecular Differentiation: Gene regulation leading to biochemical and morphological changes (e.g.,

transcription of Globin genes in RBC) More differentiated the cell, the less potential to develop into another cell type

Lyon Hypothesis X chromosome inactivation in females

Barr body Female will have same number of X chromosomes as male