1. Cell Bio Review 5 David T. Pearce Feb. 16, 2010

2. 2 Microtubule assembly Microtubules form by the addition of tubulin dimers at their ends – Three phases: nucleation, elongation and plateau – Critical concentration: balance between assembly and disassembly Addition of tubulin dimers occur more quickly at the plus ends of microtubules – Treadmilling: incorporation at plus end equals disassociation at minus end GTP hydrolysis contributes to the dynamic instability of microtubules – Dynamic instability model: presumes 2 populations of microtubules, one growing in length by continued polymerization at their plus ends and the other shrinking in length by depolarization

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4. 4 Microtubules in vivo MTs originate from microtubule-organizing centers (MTOC) within the cell – Centrosomes: composed of 2 centrioles – Pericentriolar material – The role of  -tubulin in initiation Figure 16-31b Molecular Biology of the Cell (© Garland Science 2008) Figure 16-31a Molecular Biology of the Cell (© Garland Science 2008 )

5. Centrosomes MTOCs organize and polarize the microtubules within cells – Minus ends are anchored in MTOCs – Each MTOC has a limited # of nucleation and anchorage sites – Fluctuation of pericentrin during cell cycle- highest at prophase and metaphase of mitosis 5 Figure 16-30b Molecular Biology of the Cell (© Garland Science 2008) Figure 16-30a Molecular Biology of the Cell (© Garland Sience 2008) Figure 16-29 Molecular Biology of the Cell (© Garland Science 2008)

6. 6 Microtubules in vivo-continued Microtubule stability within cells is highly regulated – Kinetochores stabilize MTs Microtubules are regulated by microtubule-associated proteins (MAPs) Motor MAPs (kinesin and dynein) – Nonmotor MAPS Tau causes MT to form tight bundles found in axons MAP2 causes MT to form looser bundle found in dendrites Figure 16-85a Molecular Biology of the Cell (© Garland Science 2008) Figure 16-85c Molecular Biology of the Cell (© Garland Science 2008) Microtubules in green Kinetochores in red Chromosomes in blue

7. Drugs can affect the assembly of microtubules Colchicine, vinblastine, vincristine and nocodazol: inhibits MT growth – Very important drugs – For example: Taxol: stabilizes MTs used in breast cancer therapy 7 Table 16-2 Molecular Biology of the Cell (© Garland Science 2008)Figure 16-23b,c Molecular Biology of the Cell (© Garland Science 2008) (B) Microtubules Untreated (C) + Taxol

8. 8 Dendritic branched networks of actin in migrating cells Wiskott Aldrich syndrome protein: WASP – Cannot produce functional WASP and therefore their platelets have difficulties undergoing changes in shape and so have difficulties in forming blood clots

9. 9 Intermediate Filaments Figure 16-20 Molecular Biology of the Cell (© Garland Science 2008) Keratin filaments in epithelial cells

10. 10 Kinesins Kinesins move along microtubules by hydrolyzing ATP Kinesins move to the “+” end of the microtubles Kinesins are a large family of proteins with varying structures and functions Figure 16-58a Molecular Biology of the Cell (© Garland Science 2008)

11. 11 Dynein Dyneins can be grouped in two major classes: axonemal and cytoplasmic dyneins Cytoplasmic dyneins move to the “-” end of the microtubules Figure 16-67 Molecular Biology of the Cell (© Garland Science 2008)

12. 12 Microtubule-based motility Cilia and flagella are common motile appendages of eukaryotic cells Cilia and flagella consist of an axoneme connected to a basal body Axoneme has characteristic “9+2” pattern with nine outer doublets of tubules and 2 additional microtubules in the center Cilia: perpendicular force Flagella: parallel force

13. 13 Basal Bodies Figure 16-84b Molecular Biology of the Cell (© Garland Science 2008) Basal Bodies Figure 16-84a Molecular Biology of the Cell (© Garland Science 2008)

14. Adult Polycytsic Kidney Disease Results from mutations in genes encoding polycystin-1 or polycystin- 2 Polycystin-1 or polycystin-2 are essential in the formation of calcium channels associated with primary cilium in the developing kidney, thus disrupting the normal mechanoreceptor function of these cilium Patients usually have multiple large cysts in both kidneys Cross section of a kidney from a patient with ADPKD showing large cysts and widespread destruction of the normal renal parenchyma 14

15. 15 Kartagener’s Syndrome The main symptom is reduced or absent mucus clearance from the lungs and thus increased susceptibility to chronic recurrent respiratory infections Many patients experience hearing loss Infertility is common in both males and females. For females, problems with ciliary action in the fallopian tubes. For males, problems with sperm motility About 50% have reversal of organs (Situs inversum) – Situs inversum : organs in body cavity are completely reversed left to right Most cases result from a mutation in the axonemal dynein arms of cilia/flagella

16. Cell migration via lamellipodia involves cycles of protrusion, attachment, translocation, and detachment Extending protrusions – Retrograde flow – Actin assembly – Rearward translocation Cell attachment – Integrins – Focal contacts Translocation and detachment – Contraction of rear and breaking adhesive contacts 16

17. 17 Receptors Tyrosine Kinase Receptor tyrosine kinases aggregate and undergo autophosphorylation The structure of receptor tyrosine kinases – Single transmembrane polypeptides – Extracellular ligand binding domain – Tyrosines on cytosolic tail of receptor – In some cases kinase activity is found in a separate protein (nonreceptor tyrosine kinase) (example: Src) The activation of receptor tyrosine kinases – Ligand binds, receptors aggregate, autophophorylation occurs between receptors – Regulatory proteins with SH2 domains recognize and bind to phosphorylated tyrosines on receptor – This binding stimulates regulatory proteins

18. 18 Activated receptor tyrosine kinases phosphorylate themselves Phosphorylated tyrosines on receptor tyrosine kinases serve as docking sites for intracellular signaling proteins Proteins with either SH2 (for SRC homology region) domains, or less commonly, PTB domains (for phosphotyrosine-binding) can bind phosphorylated tyrosines

19. 19 SH2 domains vary for specificity with neighboring amino acids The SH2 domain is a compact module. Each SH2 domain has distinct sites for recognizing phosphotyrosine and for recognizing a particular amino acid side chain, different SH2 domains recognize phosphotyrosine in the context of different flanking amino acid sequences This variation allows receptor tyrosine kinases to bind with specificity, multiple proteins containing SH2 domains

20. 20 Receptor tyrosine kinases initiate signal transduction cascade involving Ras and MAPKs Ras pathway activation – GRB2 (contains SH2 domain) interacts with tyrosine phosphorylated receptor – Sos is associated with GRB2 and becomes active Sos is a guanine-nucleotide exchange factor – Sos stimulates Ras (a monomeric G-protein) to release GDP and bind GTP. Ras is now active – Ras activates RAF (a MAPKKK) – RAF activates MEK (a MAPKK) – MEK activates ERK (a MAPK) – MAPKs phosphorylates transcription factors (such as AP-1) – Transcription factors induce to grow and divide Ras in inactivated by GTPase activating protein (GAP)

21. 21 Ras is a monomeric GTPase Ras is active when bound to GTP It is inactive when bound to GDP SOS is a GEF which “turns on” Ras GAPs “turn off” RAS Active RAS binds to RAF

22. 22 Receptor tyrosine kinases activate a variety of other signaling pathways Stimulate phospholipase C  (SH2 domain) to produce InsP3 and DAG. InsP3 stimulates calcium release. DAG stimulates PKC pathway Phosphatidylinositol-3-kinase (PI 3-Kinase) is also stimulated by receptor. Phosphatidyl inositol-3- kinase phosphorylates phospholipid in membrane. Regulates cell growth and movement

23. 23 PI 3 Kinase PI 3-Kinase produces lipid docking sites in the plasma membrane PI 3-kinase phosphorylates PI (4,5)P 2 (PIP2) into PI(3,4,5) P 3 (PIP3) Intracellular signaling proteins can interact with PIP3 via a specific interaction domain, such as pleckstrin homology domain (PH).

24. 24 AKT activation AKT and the phosphoinositol-dependent kinase (PDK1) bind PIP3 Another kinase (usually mTOR) phosphorylates AKT, this causes a conformational change that allows PDK1 to phosphorylate AKT The activated AKT now dissociates from the plasma membrane and phosphorylates various targets, including the Bad protein. Bad is involved with apoptosis.

25. 25 The PTEN tumor suppressor gene acts as a phospholipid phosphatase

26. 26 Insulin affects several signaling pathways to regulate resting glucose levels Insulin stimulates the uptake of glucose into muscle and adipose cells and by stimulating glycogen metabolism. It thereby lowers blood glucose levels The inability to produce insulin can cause type I diabetes Type II diabetes appears to result from resistance to insulin, rather than an inability to produce it. The insulin signaling pathway influences glucose homeostasis by regulating multiple pathways. – The insulin receptor is a multi-subunit receptor tyrosine kinase. – When it binds insulin, recruitment and activation of the IRS1 protein initiates signal transduction, leading to glucose import, stimulation of glycogen synthesis, and regulation of gene expression

27. 27 Insulin-Dependent Diabetes Mellitus (IDDM) (Type I Diabetes) A 28-year-old man with insulin-dependent diabetes mellitus. A, Photograph after 3 weeks of polydipsia and polyuria. B, Photograph after 5-kg weight gain with 10 days of insulin replacement. IDDM is usually caused by autoimmune destruction of islet β cells in the pancreas; this autoimmune reaction is triggered by an unknown mechanism. The destruction of islet β cells causes insulin deficiency and thereby dysregulation of anabolism and catabolism, resulting in metabolic changes similar to those observed in starvation. Among North American whites, IDDM is the second most common chronic disease of childhood, increasing in prevalence from 1 in 2500 at 5 years of age to 1 in 300 at 18 years of age. Loss of insulin reserve occurs during a few to many years. The earliest sign of abnormality is the development of islet autoantibodies when blood glucose concentrations, glucose tolerance (ability to maintain normal blood glucose levels after ingestion of sugar), and insulin responses to glucose are normal.glucose This period is followed by a phase of decreased glucose tolerance but normal fasting blood glucose concentration.glucose With continued loss of β cells, fasting hyperglycemia eventually develops but sufficient insulin is still produced to prevent ketosis; during this period, patients have non-insulin-dependent diabetes mellitus. Eventually, insulin production falls below a critical threshold, and patients become dependent on exogenous insulin supplements and have a propensity to ketoacidosis.

28. 28 Type II Diabetes Mellitus Non-Insulin-Dependent Diabetes Mellitus (NIDDM) NIDDM results from a combination of genetic susceptibility and environmental factors. NIDDM usually affects obese individuals in middle age or beyond, although an increasing number of children are becoming affected. The population risk of NIDDM is 6% to 7% in the United States. The population risk of NIDDM is 6% to 7% in the United States.

29. 29 Non-Insulin-Dependent Diabetes Mellitus (NIDDM) (Type II Diabetes) NIDDM has an insidious onset and is diagnosed usually by an elevated glucose level on routine examination. In contrast to patients with IDDM, patients with NIDDM usually do not develop ketoacidosis. In general, the development of NIDDM is divided into three clinical phases. – First, the plasma glucose concentration remains normal despite elevated blood levels of insulin, indicating that the target tissues for insulin action appear to be relatively resistant to the effects of the hormone. – Second, postprandial hyperglycemia develops despite elevated insulin concentrations. – Third, declining insulin secretion causes fasting hyperglycemia and overt diabetes. In addition to hyperglycemia, the metabolic dysregulation resulting from islet β-cell dysfunction and insulin resistance causes atherosclerosis, peripheral neuropathy, renal disease, cataracts, and retinopathy. One in six patients with NIDDM will develop end-stage renal disease or will require a lower extremity amputation for severe vascular disease; one in five will become legally blind from retinopathy. Chronic hyperglycemia can be monitored by means of measurements of the percentage of hemoglobin that has become modified by glycosylation, referred to as HbA1c. – Rigorous control of blood glucose levels, as determined by HbA1c levels as close to normal as possible (<7%), reduces the risk of complications by 35% to 75% and can extend the average life expectancy, which now averages 17 years after diagnosis, by a few years.

30. Insulin Pathway – The Simple Overview SIGMA-ALDRICH

31. Insulin Signaling The Slightly Complex Overview 31 Image from http://www.nature.com/nature/journal/v414/n6865/fig_tab/414799a_F2.html

32. 32 The insulin receptor–AKT signaling pathway Activation of AKT (PKB) has four important consequences First, it leads to movement of GLUT4 from vesicles in the cytosol to the plasma membrane, allowing glucose uptake. Second, AKT can phosphorylate a protein known as glycogen synthase kinase-3 (GSK-3), reducing its activity. This leads to an increase in glycogen synthesis Third, it alters TSC/mTOR signaling pathway and thus protein translation Fourth, it alters FOXO pathway leading to deceased glucose synthesis

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34. Role of TSC1 & 2 in the mTOR signaling Tuberous sclerosis complex (TSC) tumor suppressors (TSC1 and TSC2) is regulated by multi-site phosphorylation and acts as a point of integration for cellular signaling When active, the TSC1-TSC2 complex acts as a GTPase activating protein (GAP) for the Ras- related small G protein Rheb. – Thus turning Rheb off by stimulating its intrinsic GTPase activity. In the presence of growth factors and nutrients, this TSC1-TSC2 complex is turned off, allowing the GTP-bound active version of Rheb to accumulate and turn on downstream pathways such as mTORC1 The mTORC1 complex is a critical regulator of cell growth and proliferation. 34

35. Tuberous sclerosis Tuberous sclerosis is an inherited disorder whose key features include multiple facial angiofibromas, hypopigmented macules, periungual fibromas, seizures, Shagreen patch, cardiac rhabdomyoma, and renal lesions. Many patients also have ocular and neurologic manifestations, including mental retardation. The disorder results from a mutation in either TSC1 or TSC2 ~Two-thirds of cases may be due to spontaneous genetic mutations. TSC2 mutations account for the majority of sporadic cases – these individuals usually show a more severe phenotype that includes renal lesions and neurologic deficits 35 Multiple facial angiofibromas

36. 36 Example of a dominant negative disorder: Achondroplasia Achondroplasia also known as short-limbed dwarfism; charaterized by small stature with short limb, large head, low nasal bridge, prominent forehead, lumber lordosis. – Incidence is ~ 1 in 10,000 Pathology – Failure of cartilage cell proliferation at the epiphyseal plates of the long bones, resulting in failure of longitudinal bone growth, causing short limb. Mutation in Fibroblast Growth Factor Receptor 3 (FGFR3) gene; – ~ 80% of patients are due to new mutation; – increased risk with late paternal age

37. 37 Achondroplasia, an autosomal dominant disorder that often occurs as a new mutation. Note small stature with short limbs, large head, low nasal bridge, prominent forehead, and lumbar lordosis in this typical presentation. Achondroplasia is a well known, incompletely dominant skeletal disorder of short-limbed dwarfism and large head Most achondroplastic individuals have normal intelligence and lead normal lives within their physical capabilities. Marriages between two achondroplastic individuals are not uncommon.

38. 38 Some growth factors transduce signals via receptor serine/threonine kinase Transforming Growth Factor Beta receptor is an example of serine/threonine kinase – TGF ligand binds a complex of type I and II receptors – Type II receptor phosphorylates type I receptors – Type I receptors phosphorylates an R-Smad (Smad2 or Smad3) – R-Smad complexes with Smad4 – In the nucleus, the Smad complex associates with transcription factors to modulate gene expression

39. Signaling Pathway of TGF-  SIGMA-ALDRICH

40. 40 JAK-STAT Signaling Pathway Cytokine receptors activate the JAK-STAT signaling pathway, providing a fast track to the nucleus Tyrosine kinase-associated receptors called JAK-STAT are often used by cytokines to regulate the proliferation of certain cells involved in the immune response. The receptor itself has no intrinsic kinase activity, but it binds the tyrosine kinase JAK (Janus kinase) STAT proteins are located in the cytosol and are referred to as latent gene regulatory proteins because they only migrate into the nucleus and regulate transcription after they are activated There are many different STAT proteins. – Receptors for different cytokines bind different STATs

41. 41 JAK-STAT Signaling Pathway

42. 42 The receptor protein Notch is a latent gene regulatory protein Notch signaling is very important during development For example, when individual epithelial cells begin to develop as neural cells, they signal to their neighbors not to do the same through Delta binding receptor Notch Upon binding Delta, Notch is cleaved on either side of the plasma membrane The freed cytoplasmic tail of Notch then migrates to the nucleus, associates with other regulatory factors bound to promoter elements and modulates gene transcription

43. 43 The Wnt/  -catenin signaling pathway Wnt proteins are secreted signal molecules that act as local mediators and morphogens to control many aspects of developments. Wnt protein bind to Frizzled receptors and inhibit the degradation of  -catenin APC is a key protein in the regulation of  -catenin – APC is often mutated in some forms of colon cancer  -catenin accumulates and translocates to the nucleus Once in the nucleus,  -catenin migrates to the nucleus, displaces Groucho and associates with coactivator

44. 44 NF-  B-Dependent Signaling Pathway The NF-kB proteins are latent gene regulatory proteins that are present in most animal cells and are central to many stressful, inflammatory, and innate immune responses. These responses occur as a reaction to infection or injury and help protect stressed multicellular organisms and their cells. Upon cell signaling, the inhibitory protein (IkB) dissociates from NF-kB in the cytosol. NF-kB migrates to the nucleus, where it interacts with coactivators to alter gene transcription