Cell Biology - Class Notes for Exam #3

• Sorting Pathways and Exocytosis
• Endocytosis
• ER, Golgi and Sorting Signals
• Cell Cycle and Cancer
• HIV-1 and AIDS
• Immune System (separate page)

LSc 202 Sorting Pathways and Exocytosis Notes Winter 1999

Chapter 20 pp. 855-863 text

Sorting pathway examples:

cytosolic proteins

organelle-bound (ER membrane)

secretory – ECM components, soluble mediators

plasma membrane proteins

Addresses: signals for directing to final destination are found within the protein

exocytosis and endocytosis use same pathway à mechanisms for simultaneous transport & distribution of proteins traveling in opposite directions

Exocytosis – default pathway for ER-directed proteins w/ no further signal sequences

Transcytosis à endocytosis on one side of the cell membrane – sort immediately to secretory vesivles which fuse with the pl. membrane on opposite side of cell

Machinery à microtubule tracks

Examples of secreted proteins: digestive enzymes, mucus, hormones

Constitutive vs Regulated mode of secretion

Constitutive (presumed to be the default pathway): continuous release (on all the time) Ex. mucus, collagen; exocytic vesicles have about 2X the concentration of the secreted protein as the trans Golgi cisternae

Regulated: released at intervals upon signal, Ex: neurotransmitters, some hormones; proteins stored in storage secretory vesicles which concentrate the proteins to be secreted 100X trans Golgi

Signals from outside cell à 2^nd messenger pathways are involed, Ex: increase in intracellular calcium is observed before insulin release; other pathways involve cAMP or GTP; protein kinases willl activate fusion-promoting proteins for rapid release of vesicular contents

Same cell can have both pathways: Ex: pituitary gland ACTH (peptide hormone) regulated – laminin release is constitutive

Polarized – release on one side of cell one set of proteins, different set release on other side Ex: small intestine epithelial cells -- digestive enzymes released on intestinal cavity surface/ECM componenets released on side facing interior of body
Ex: neurons: vesicle release only at axon terminals

LSc 202 Endocytosis Notes Winter 1999

Endocytosis: substances passing into the cell, cell engulfs/ingests material and fluid from surroundings

• Phagocytosis (cell eating): cells engulf large particles (Ex: bacteria); receptor-bound large insoluble aggregates of molecules, cell parts, whole cells
• Pinocytosis (cell drinking): cells engulf extracellular fluids & dissolved materials: bulk-phase (non-specific) engulfs whatever particles are in surrounding medium
• Receptor-Mediated Endocytosis (RME): a type of pinocytosis where specific substances are selectively internalized via binding to a cell surface receptor molecule

Steps in RME:

1. Binding of substrate to specific cell surface receptor
2. Formation of coated pit
3. Formation of coated vesicle
4. Vesicle movement and sorting of vesicular contents
5. Recycling of the receptor (some receptors are degraded)
6. Fusion with Golgi or fusion with lysosomes

Coated Pits: clathrin

• basket-like assembly composed of polypeptides forming regular geometric patterns
• coat is disassembled after the vesicle has pinched off and is free in cytoplasm
• major component clathrin, clathrin polypeptide subunit forms a triskelion (3 bent legs), bead-like swellings on ends (Fig.20-31)
• slight bending of legs can form pentagon, hexagon & other lattices
• adaptors link clathrin to receptors
• may function to reinforce pl. membrane on cytoplasmic side during endocytosis
• may provide energy for invagination
• coats assemble without input of cellular energy
• disassembly requires an "uncoating protein" with ATPase activity
• clathrin is also associated with trans Golgi vesicles (but not others)

pH and endocytosis

• endocytic vesicle at membrane (near neutral pH 7.0)
• endocytic vesicle in cytoplasm (early endosome – pH drops to 5 - 5.5)
• lysosomes (pH 4.5 - 5), contain hydrolytic enzymes; degrade materials by fusion with endosomes, or autophagy – degrades engulfed organelles
• ligand release by receptors is often pH dependent
• Ex: LDL receptor releases ligand at acidic pH and receptor recycles
• Ex: transferrin receptor remains bound to transferrin at acidic pH, but releases iron at acidic pH, and recycles
• Ex: EGF receptor remains bound to EGF at acidic pH and is degraded with ligand in lysosomes
• CURL (compartment of uncoupling of receptor from ligand)

LSc 202 ER, Golgi, and Sorting Signals Notes Winter 1999

ER structure (Fig. 20-3): lumen (inside stacks or cisternae), there are some direct connections between outer nuclear membrane and ER (perinuclear compartment) Fig. 20-4

Enzymes in ER lumen and ER membrane (and some in smooth ER)
RER

• enzymes which add initial stem structure of carbohydrates
• enzymes which assemble polypeptide subunits (Ex: help form disulfide linkages)
• enzymes important in lipid synthesis

RER and/or SER

• enzymes important in initial reactions in fat oxidation
• enzymes important in glycogen metabolism
• enzymes important in steroid hormone synthesis
• enzymes important in detox

Golgi (Figs. 20-6, 20-5): cis vs trans Golgi cisternae

Enzymes in Golgi (unequally distributed in cisternae -- Table 20-1)

• enzymes which add or remove sugars
• enzymes which add sulfate, acetyl, and phosphate groups
• enzymes which add fatty acids to proteins
• enzymes which remove terminal amino acids or interior amino acids

________________________________

Microtubles of the cytoskeleton are important for maintaining ER and Golgi juxtaposition within the cytoplasm

Path of new proteins: ER ® cis Golgi® trans Golgi ® elsewhere (Fig 20-9)

Pulse-Chase experiments: ³H-leucine Fig 20-10

3 minute pulse ® RER labels

3 minute pulse, 17 minutes chase ® RER, Golgi, and some secretory vesicles label

3 minute pulse, 117 minute chase ® secretory vesicles label

Sorting Signals

Signals encoded in the protein sequence

• N-terminal signal sequence: for ER insertion also found for insertion into mitochondria and chloroplasts
  • usually clipped once inside the organelle (by enzyme signal peptidase)
  • combination of polar and nonpolar residues (many hydrophobic)
• interior signal sequence: for nuclear targeting (signal depends on the chemical properties of the sequence, not the actual sequence
• C-terminal signals: targets to microbodies
• Signals beyond the initial ER targeting sequence
  • targeting to internal compartments (other organelles)
  • targeting to membranes of organelles
  • ER retention signals: KDEL (lys-asp-glu-leu)
  • targeting to lysosomes: Mannose-6-P

 

Signal Hypothesis (1975)

• mRNAs for proteins synthesized on RER contain sequnces at the beginning for ER insertion
• stop-transfer signal would keep protein stuck in the ER membrane

SRP (signal recognition particle)

• allows ribosomal attachment to ER
• large complex of 6 polypeptides + RNA
• binds the N-terminal ER insertion signal sequence
• Fig 20-17

Co-translational vs Post-translational ER insertion

• co-translational (more common) occurs with signal sequence
• post-translational
  • requires ATP
  • chaperones (stabilize emtering proteins in an "unfolded" state)

LSc 202 Cell Cycle and Cancer Notes Winter 1999

The Search for Molecular Switches

mutants in which cell cycle is altered or arrested give genetic basis for cell cycle regulation

Cell cycle: time required varies widely among cell type and species (the length of G₁ is typically what determines the length of cell cycle)

• 10 hr – intestinal epithelium
• some cells – days, months, years…
• 15-20 min. most rapid – early embryonic divisions in some animals (essentially negligible G₁)
• some cancer cells – less than 1 hr
• some cells stop dividing completely when differentiation is complete (Ex: some cells of CNS and muscle tissue in mammals)

Arresting cells in part of G₁ (G₀)

• deprivation of nutrients
• inhibitors of RNA or protein synthesis
• contact inhibition
• few examples of cells arresting in G₂ with duplicated chromosomes

 

Cell growth is controlled by:

1. signals from the cell cytoplasm
2. external hormones (Ex. steroid hormones), growth factors (PDGF, EGF); mediated through cell surface receptors and protein kinase activity (either directly by receptor or through 2^nd messenger pathways)
3. contact (density-dependent) inhibition – dependent on surface glycoproteins; contact inhibited cells will often produce and secrete negative growth signals (Ex: interferons)
4. changes in ion transport induced by surface binding of growth mediators
5. pH – cytoplasmic pH rises 0.2-0.4 units during late G₁ and falls before S
6. histone and non-histone protein modifications (Ex: phosphorylation of non-histone proteins G₁ à S transition)
7. polyamines (multiple amino groups giving positive charge at cellular pH), formed by enzyme converting a.a. arginine, linked to cAMP and modulated by phosphorylation; increased polyamines speed entry into S phase, inhibition of polyamines causes cells to arrest in S or G₂

Cancer: uncontrolled cell division (no longer contact inhibited)

These out-of-control cells can form tumors

• benign tumors: fail to grow & spread, rarely cause problems (do not metastasize)
• "cancer" malignant tumors: continue to grow & often spread to other parts of the body (forming secondary tumors)
  metastasis = spread, spread through the circulatory and lymphatic systems

Characteristics of Cancer Cells

• cells divide faster than normal cells from which they are derived
• tumor cells are de-differentiated (altered surface properties, lose some characteristics of cells they are derived from)
• tumor cells produce progeny identical to themselves
• heritable
• transplantable
• lack contact inhibition
• can infiltrate nearby tissues (attach to basal laminae, penetrate tissue to bloodstream

How does cancer start?

Normal cell ð Tumor cell (transformation)

• conversion – often begins with a mutation in DNA (caused by chemical, physical, or biological events)
• carcinogen – cancer-causing agent (e.g. UV radiation, cigarette smoke, X-rays, over 1000 known chemicals including some pesticides, household cleaners, food additives), act by causing alterations in DNA
• not all mutations cause cancer: mutation in growth control genes sometimes can release cells from the normal growth control (no contact inhibition)
• oncogenes – genes arising from normal cellular genes, which when mutated cause a loss of growth control – cancer. These genes wither change their level of activity or produce proteins with altered amino acid sequences.

  Some types of genes which can be converted to oncogenes encode for:

  • growth factors
  • receptors for growth factors
  • regulatory proteins involved in cell growth (nuclear transcription factors)
  • protein kinases (enzymes which phosphorylate other proteins)
  • G proteins

  How are proto-oncogenes converted to oncogenes?

  • sequence changes in genes or control regions of genes
  • deletions from genes or control regions of genes
  • translocations of genes Ex: Philadelphia chromosome: translocation of 9 à 22 causes chronic myeloid leukemia by placing the abl gene (Abelson oncogene) by the bcr gene (breakpoint cluster region); the resulting fusion protein abl-bcr that is translated from the 2 genes is an active oncogene
  • amplification of gene à extra copies

• tumor suppressor – genes which normally inhibit cell growth, "recessive" cancer-causing mutations in that both copies of gene must be mutated to produce effect
  • Ex: Retinoblastoma (RB gene) rare childhood eye cancer caused by 2 mutations in the RB gene – usually one inherited germline mutation and one somatic (environmentally caused) mutation. The producte of the RB gene normally binds transcription factors so they can’t activate certain genes, inhibiting cell division
  • Ex: p53 normally encodes a transcription factor, prone to point mutations in a 600 bp region, there have been distinct mutations found in p53 in a variety of cancers (colon, liver, breast, bladder, lung, blood, brain, esophagus, skin)
    p53 may be considered a genetic mediator between environmental insults and the development of cancers; most p53 mutations are somatic
    Rare familial p53 germline mutation (Li Fraumeni family cancer syndrome)
  • Both p53 and RB have been implicated in a variety of cancer types suggesting that lifting of tumor suppression may be a common factor in many cancers
• After the initial mutation it may take many years before the cancerous tumor emerges (20-30 years)

Review:

• Single somatic mutation cancers (dominant) à oncogenes which activate genes that promote cell growth
• Inherited predisposition (recessive) à inherit a mutation in (constitutional) germline sequence of one copy of gene; a second somatic mutation triggers uncontrolled cell growth (Ex. tumor suppressors)
• Inherited cancer à disorders in DNA replication (Ex: xeroderma pigmentosum)

 

Multi-step progression – often multiple alterations over a period of years is necessary for full malignancy (Fig 22-21)

Viruses and Cancer: ~20% tumors are caused or promoted by viruses

• retroviruses – incorporate RNA genome into host DNA, occasionally these retroviruses contain genes capable of causing uncontrolled cell growth
• DNA tumor viruses (Table 22-7) – viral genes encode uncontrolled cell growth
  • common viral groups infecting humans
  • some cause benign growths Ex: some papilloma viruses cause relatively harmless skin and veneral warts, others are implicated in cervical carcinoma

Treatments:

• surgery: tumors can be surgically removed
• radiation: tumors cells can be destroyed by targeted radiation
• chemotherapy: highly toxic agents which kill rapidly dividing cells of secondary tumors; can cause serious side effects (nausea, chills, hair loss, general sickness)
• combinations of surgery, chemotherapy, radiation
• immunotherapy: antibody therapy directed toward tumor specific antigens
• gene therapy: using DNA to target specific cells to die

Prevention of Cancer:

• What types of things will you do in your life to avoid cancer?
  • don’t smoke
  • avoid UV and radon exposure
  • avoidance of excessive dietary carcinogens (Table 22-9) – cultural differences in frequencies of certain cancers may be related to diet! (stomach cancer is high breast cancer low in Japan)
  • awareness of early warning symptoms
  • regular physical exams (both self and physician)
• Would a family history of cancer make you more aware of these things?
• Warning signs
  • bowel or bladder changes
  • sore that doesn’t heal
  • bleeding or discharge
  • lump
  • chronic indigestion
  • difficulty swallowing
  • wart /mole change in appearance
  • chronic cough/hoarseness

LSc 202 HIV-1 and AIDS Notes Winter 1999

1981 first cases in US, 1984 "discovered"

Retroviruses ® RNA viruses

Fig 19-1: Components of HIV-1

• core -- RNA and enzymes
• protein coat
• membrane or envelope (host-derived)
• spikes (gp41 anchor and gp120 extends from membrane; primary receptor for viral entry)

Host cell receptor" CD4 molecule (primarily found on T helper cells and some macrophages)

Course of HIV infection:

• free virus particles enter host system
• gp120 binds CD4
  Coreceptors:
  CCR5 -- important in early infection, macrophage population
  CXCR-4 -- important later in HIV, T cell population
• membrane fusion
• injection of viral core
• viral RNA converted to DNA by enzyme reverse transcriptase (RT)
• viral DNA integrated into host genome (enzyme integrase)
• viral genome transcription and translation
  |retroviruses have gag-pol-env genes
  gag -- core proteins associated with RNA
  pol -- enzymes (RT and integrase)
  env -- glycoproteins of the coat
• packaging of virions
• budding from host cell of new infectious virions

Clinical Problem with HIV -- steady destruction of helper T cells, which in effect disables both arms of the immune system (humoral and cellular) see Overhead Handout

Clinical Stages of HIV infection and AIDS

• initial (3-5 weeks) and symptomless period (3-10 years)

  flu-like symptoms in first few weeks

  sero-conversion -- blood contains anti-HIV antibodies (HIV⁺) usually in within first 6 months or less

• AIDS - related complex -- when T_Helper cell count is 200-400/mL blood; normal T_Helper cell count is ~1000/mL blood

  patients typically experience mild afflications that can persist (Ex: fungal infections like athlete’s foot, herpes outbreaks etc)

• full blown AIDS -- when T_Helper cell count is less than 200/mL blood

  patients are prone to opportunistic infections:

  Ex: cytomegalovirus (CMV), pneumocystis carinii, TB, toxoplasma, mycobacterium, Kaposi’s sarcoma, several disorders causing memory loss and dementia

  Assignment: Read Journal Article and pick one vaccine strategy to learn in detail.

Letvin, Norman L. (1998) Progress in the development of an HIV-1 vaccine. Science 280:1875-1880.

Discussion of Article:

What does a vaccine need to do?

• in general vaccines are designed to elicit an immune response involving antibodies (humoral response) and create immunological memory in order to combat the foreign antigen upon future exposure and create neutralizing antibodies which prevent viral entry into host cells
• Most current effective vaccines produce antibody responses not cellular effector T cells
• For HIV-1 -- a vaccine needs to create a memory humoral response which generated neutralizing antobodies and create an HIV-1 specific CTL (cytotoxic or killer T lymphocyte) response

Why is HIV-1 a tough target?

• HIV-1 does elicit humoral and cellular immune responses, but anti-HIV antibodies in patient sera show a poor neutralizing ability and don not seems to prevent viral entry into host cells
• HIV-1 repidly mutates during infectious stages and escape future immune detection
• HIV-1 persists indefinitely as latent proviral DNA
• due to HIV-1 sexual transmission through mucosal surfaces -- need to establish mucosal immunity which is problematic

Studying patient responses

• serum antibodies in general are poor neutralizers, mainly recognize gp120 epitopes (places on the gp120 protein that are antigenic)
• some monoclonal antibodies have been isolated which neutralize viral entry, these antibodies recognize both gp120/gp41 together
• HIV-1 specific CTLs (CD8⁺ T killer cells) can inhibit replication of HIV-1 in CD4⁺ cells via lysis or secretion of inhibitory chemokines and cytokines
• early containment of virus coincides with virus-specific CTLs
• increased CTL correlates with decreased viral load and stable clinical status

 

Animal Models

Summarized in Table-1 or article p. 1876

Strategies for vaccine design

vaccines must be safe, several strategies involve deactivated non-pathogenic forms of HIV-1, the fear is that these inactivated forms will mutate or evolve such that they regain their pathogenicity

Summarized in Table-2 of article p. 1877

Link to Immune System Notes Page