Exocytosis and endocytosis

2. Endocytosis Why do cells need endocytosis? Is there more than one endocytic pathway ? Clathrin-mediated uptake Caveolae Non-clathrin/non-caveolae pathways Pinocytosis/ Phagocytosis What are the functional consequences of endocytosis? How are endocytic structures formed and how do they know where to go? Where do the textbook models come from?

3. Endocytosis Why do cells need endocytosis? Is there more than one endocytic pathway ? Clathrin-mediated uptake Caveolae Non-clathrin/non-caveolae pathways Pinocytosis/ Phagocytosis What are the functional consequences of endocytosis? How are endocytic structures formed and how do they know where to go? Where do the textbook models come from?

4. Getting molecules into cells: crossing the plasma membrane Diffusion across the plasma membrane water, gases, small molecules Protein-mediated transport ion channels, transporters Pore formation toxins Membrane fusion viruses Formation and internalization of membrane-limited vesicles Endocytosis Pinocytosis Phagocytosis

5. Endocytosis Small region of the plasma membrane invaginates to form membrane-limited vesicles (0.05-0.1 mm diameter) Internalized molecules retain topology (lumenal = extracellular) Cargo can be specifically selected Destination of cargo can be controlled; destination depends on pathway of uptake Cargo receptors can be recycled to the cell surface Conditions under which internalization occurs can be regulated

6. Endocytosis Why do cells need endocytosis? Is there more than one endocytic pathway ? Clathrin-mediated uptake Caveolae Non-clathrin/non-caveolae pathways Pinocytosis/ Phagocytosis What are the functional consequences of endocytosis? How are endocytic structures formed and how do they know where to go? Where do the textbook models come from?

8. 1. Clathrin-mediated endocytosis

9. Compartments and pathways in clathrin-mediated endocytosis

10. 2. Caveolae

11. Caveolar endocytosis

12. Caveolar endocytosis Caveolae are 50-100 nm invaginations on the cells surface Caveolin, a membrane protein, is the �coat protein� of caveolae Do not undergo constitutive endocytosis but can undergo endocytosis in response to a signal (ex. SV40 binding) in a cholesterol- and dynamin-dependent fashion Internalized caveolae recruit actin to form �comet tails� Upon internalization caveolae are delivered to novel endosomal compartments known as �caveosomes�

13. 3. Non-clathrin/non-caveolae Currently poorly understood Defined by process of elimination Multiple mechanisms for testing for clathrin-mediated uptake mechanisms Both caveolae and clathrin-mediated endocytosis involves dynamin Caveolae endocytosis can be inhibited by dominant negative caveolin Caveolae endocytosis is inhibited by cholesterol depletion Emerging theme: involves detergent-resistant membranes/ lipid rafts

14. Lipid rafts Membrane microdomains enriched in glycolipids and cholesterol Function by segregation: some proteins are enriched in rafts, others are excluded Participate in TGN-to-PM trafficking of apically-destined proteins Function at the PM in endocytosis and in organizing cell signaling pathways Controversial model

15. 4. Pinocytosis/ phagocytosis Pinocytosis: internalization of fluid Actin dependent Generated at sites of ruffling at the plasma membrane Includes macropinocytosis (vesicles > 1 mm in diameter) and micropinocytosis (vesicles < 200nm in diameter) Phagocytosis: internalization of particles Occurs in specialized cells ex. neutrophils and macrophages Actin dependent Clathrin independent Particles > 0.5 mm in diameter

16. Phagocytosis vs. clathrin-mediated endocytosis

17. How can you tell if endocytosis is clathrin mediated? Should be inhibited by� Overexpression of mutant forms of coat proteins- dominant negatives (DN) interfere with function of normal proteins DN Dynamin K44A (also inhibits caveolae) DN Eps15- binds AP-2, arrests coat assembly Clathrin hub domain overexpression Expression of m2 subunit of AP-2 (affects YXX� but not di-leucine containing proteins) Potassium depletion or incubation in hypertonic medium- interfere with clathrin coat assembly Typically use a marker for this pathway (ex. transferrin) as a positive control in conjunction with these treatments to show they are effective

18. How can various endocytic routes be further differentiated experimentally?

19. Endocytosis Why do cells need endocytosis? Is there more than one endocytic pathway ? Clathrin-mediated uptake Caveolae Non-clathrin/non-caveolae pathways Pinocytosis/ Phagocytosis What are the functional consequences of endocytosis? How are endocytic structures formed and how do they know where to go? Where do the textbook models come from?

20. Some of the functions of endocytosis Nutrient uptake Receptor recycling Plasma membrane protein downregulation and/or degradation Synaptic vesicle recycling Transcellular signaling Exploitation: virus and toxin entry into cells

21. Low density lipoprotein (LDL) particles are a carrier for cholesterol LDL particles are taken up by clathrin-mediated endocytosis Mutant LDL receptors in patients with familial hypercholesterolemia are defective in internalization ex. tyrosine to cysteine change in YXX� motif in cytosolic domain The ligand and receptor separate in late endosomes due to low pH The LDL receptor is recycled to the cell surface The LDL particle is broken down in lysosomes

22. Transferrin is a glycoprotein that binds and transports iron Apotransferrin = iron-free Ferrotransferrin = two bound iron atoms Transferrin receptors bind ferrotransferrin at neutral pH At low pH (of late endosomes), iron releases from transferrin while apotransferrin remains bound to the receptor Apotransferrin and the transferrin receptor are recycled to the cell surface where apotransferrin is released

23. Removal of activated receptors from the cell surface i.e. receptor downregulation Ligand binding induces receptor internalization Targets receptors for degradation Some receptors can continue to signal until they are incorporated into multivesicular bodies

24. Some viruses like influenza exploit the low pH in endosomes as a fusion trigger

25. Endocytosis Why do cells need endocytosis? Is there more than one endocytic pathway ? Clathrin-mediated uptake Caveolae Non-clathrin/non-caveolae pathways Pinocytosis/ Phagocytosis What are the functional consequences of endocytosis? How are endocytic structures formed and how do they know where to go? Where do the textbook models come from?

28. Sorting signal for clathrin-mediated endocytosis AP-2 is the PM clathrin adaptor Sorting signals for incorporation into clathrin coated pits include di-leucine motifs and YXX� Many other regulatory proteins involved in clathrin-mediated endocytosis Sorting signals into non-clathrin endocytic pathways are not well understood

29. Protein-protein interactions that suggest a link between endocytic and cytoskeletal machinery Actin filaments faciliate clathrin-mediated endocytosis under some conditions but disruption of filaments does not universally inhibit vesicle formation in higher eukaryotes Genetic evidence in yeast indicates a link between actin and endocytosis Actin tails associate with some endocytic vesicles

30. Endocytosis Why do cells need endocytosis? Is there more than one endocytic pathway ? Clathrin-mediated uptake Caveolae Non-clathrin/non-caveolae pathways Pinocytosis/ Phagocytosis What are the functional consequences of endocytosis? How are endocytic structures formed and how do they know where to go? Where do the textbook models come from?

31. Example from the literature:defining a clathrin-independent internalization pathway How does cholera toxin get inside cells?

32. Cholera toxin modifies Gsa subunits and constitutively activates adenylyl cyclase Cholera toxin binds to and enters intestinal cells Once inside the cell, it covalently modifies a heterotrimeric G-protein Leads to persistant adenylyl cyclase activation, elevation of cAMP, and chloride secretion Causes diarrhea

33. How does cholera toxin (CTX) get to the right place to do its dirty work?

34. Endocytosis and retrograde transport of bacterial protein toxins

35. Is cholera toxin internalized to the Golgi complex by a clathrin-dependent process? Epsin and eps15 mutants inhibit clathrin-mediated transferrin (Tf) uptake to recycling endosomes Epsin and eps15 mutants do not affect cholera toxin B-subunit (CTXB) uptake to the Golgi complex (marked by b-COP) Suggests CTXB is delivered to the Golgi complex by a clathrin-independent pathway

36. Does internalized CTXB pass through early endosomes? Early endosome function requires the GTPase Rab5 Dominant negative rab5 S34N (GDP bound) expression perturbs early endosomes and blocks transferrin uptake Rab5 S34N does not affect delivery of CTXB to the Golgi complex Suggests CTXB does not pass through early endosomes

37. Current questions What are the physiological cargo carried by non-clathrin mediated endocytic pathways? How does cholera toxin get targeted into caveolae/ lipid rafts? What role does caveolin play in the internalization of cholera toxin? Do clathrin-mediated and non-clathrin mediated endocytic pathways intersect? What cellular machinery is responsible for non-clathrin pathways?