Secretory Systems and Polarity PDF

Secretory Systems and Polarity Apical vs Basolateral Axonal vs Dendrite What will we learn today • Vectorial sorting and selective retention are keys to polarization of membranes • Many different sorting signals used in cells – Sorting signals can be present either in the cytoplasm or the lumen •...

Secretory Systems and Polarity Apical vs Basolateral Axonal vs Dendrite What will we learn today • Vectorial sorting and selective retention are keys to polarization of membranes • Many different sorting signals used in cells – Sorting signals can be present either in the cytoplasm or the lumen • Microtubules are important in differentiation of neuronal processes Epithelial cells have two distinct plasma membranes • Some cells have distinct environments on either side of the cell (e.g. kidney cells) – Apical side faces the outside world; often hostile environments (e.g. stomach acids). – Basolateral side faces the inside world; neighboring cells and internal tissues • Plasma membrane and secreted proteins are distinct to face different environments. – Ion channels; transporters, lipids must be distinctly targeted to maintain correct balance. Tight junction defines interface between apical and basolateral sides • Tight junctions made of transmembrane proteins tightly linked to the cytoskeleton prevent leakage through epithelial layer and prevent diffusion of membrane Two general solutions • Vectorial sorting: components of apical and basolateral membranes are sorted into distinct vesicles in the TGN or in endosomes that then fuse specifically to their target membranes. • Selective retention: After fusion, proteins are endocytosed and reinserted, but are not endocytosed from the correct compartment • Can combine two mechanisms. • Hard to generalize; different for distinct proteins in same cell; different for same protein in different cells. VECTORIAL SORTING Red Arrows indicate direct transport from the TGN to either the apical or basolateral compartment. Blue arrows represent initial sorting from the TGN into recycling endosomes (RE). Some proteins (green arrows) are initially targeted the wrong Schuck, S. et al. J Cell Sci 2004;117:5955-5964 compartment (i.e. basolateral), but are Basolateral sorting • Direct sorting in TGN into vesicle that is directed to basolateral plasma membrane. • Sorting to common endosome and then into vesicle that is specific for basolateral plasma membrane • Non-specifically sorted to plasma membrane and then endocytosed to recycling endosome and then into vesicle that is specific for basolateral plasma membrane Basolateral signals are in cytoplasm • Tyrosine-based motif (YXXwhere is a bulky hydrophobic group such as phenylalanine or tryptophan (Binds to mu subunit of AP1 and AP2s). • Di-leucine motif (D/ExxxLL) or (RxxxLL) or (LLxxxD/ED/E). (Binds to beta subunit of Adapter proteins AP1 and AP2 • These signals are similar to endosome and endocytosis signals as common binding to adaptor subunits such as AP1 and AP2. cytoplasm lumen AP1B subunit specific for polarized cells • Special subunit of AP-1 only found in polarized cells (AP1B) that contains different m subunit (m1B). The m subunit binds to cargo. – AP1B does not co-localize with AP1. AP1 is at TGN, but AP1B is at recycling endosomes, so works in step from recycling endosome to basolateral membrane – AP1B only important for basolateral proteins with tyrosine-based motif. • In cells lacking this molecule, proteins with tyrosine sorting signals are mis-sorted but not AP1B proteins with dileucine based signals Apical sorting • Directly through transport vesicles. • Sorting through endosomes • Sorting through basolateral membranes and then transcytosis through endosomes. Apical signals are usually found in the membrane or the lumen • Lipid linkage (Glycosylphosphatidylinositol; GPI) • Transmembrane domain • Glycosylation signals (O-glycosylation; N-glycans); aggregation by co-factors • Segregation into rafts by aggregation of proteins. • Exceptions (Syntaxin-III) sorting signal is cytoplasmic in cytoplasmic domain lumenal Raft clustering and domain-induced budding Black proteins are apical targeted proteins. The black proteins are aggregated by a lumenal adaptor and then binds to the membranes. There is also lipid segregation (gray rafts) and aggregation. This causes budding. There is no known coat on apicalSchuck, S. et al. J Cell Sci 2004;117:5955-5964 transported structures and they Two distinct types of apical transport vesicles • In the same cell, two different apically transported proteins are found in two distinct vesicles – In same exit vesicle from TGN, sorting happens in endosomes. – One aggregates with annexin in lipid rafts – The other aggregates with a leptin, galectin 3 – Both these sorting/aggregation SNAREs distinguish vectorial routes • Basolateral transport, but not apical transport was blocked by tetanus toxin (Toxin works by cleaving V-SNARES involved in fusion). – V-snare used for apical sorting not sensitive to toxin (TI-VAMP) – Cellubrevin implicated in Ap1B vesicle fusion • Different types of apical transport sensitive to different V-SNAREs as well – Direct route blocked with TI-VAMP; Indirect route through transcytosis uses VAMP8 Neuronal polarity • Neurons have more polarity than other cells • Neurons are very complicated cells with multiple unique compartments • Axon vs dendrite differentiation is a modification of apical (axon) vs basal (dendrite) polarity Neuronal Organization Dendrites (information input) Axon Hillock (site of action potential generation Pre-synaptic Terminal Axon (information output) Spines (Inputs) Nucleus Site of Transcription Cell Body Site of most Protein synthesis and Vesicular sorting Axon determination in culture Axonal determination is a good model for understanding how polarity is generated. Axon Determination Rules • Longest neurite becomes axon – May be determined by last cell division by placement of centrosome and golgi apparatus. • Neurite with greatest amounts of dynamic actin becomes axon – Can induce multiple axons with cytochalasin D (a drug that destabilizes actin filaments) to induce actin instability. – Can make any neurite an axon by local application of cytochalasin D • Cutting an axon shorter than another neurite allows other neurite to become axon. Motor proteins (Kinesin) are important • To sort vesicles down axons and dendrites need motor proteins to move them on microtubules. • A specific isoform of kinesin only transports vesicles down axons, not dendrites. • Can use this as a marker of axonal determination, but it is also a mechanism for axonal determination. Live imaging of Kinesin How does Kinesin know which process is the axon? • Signaling – PI-3 kinase signaling at tip of process is necessary and sufficient to determine the axon • Actin and microtubule cytoskeletal dynamics are downstream of this pathway • Both Positive and negative feedback mechanisms are important for only one neurite becoming an axon How does Kinesin know which process is the axon? • Dynamic microtubules – Kinesin is a motor that travels down microtubules – The kinesin isoform going down axons recognizes post-translational modifications on microtubules (prefers detyrosinated microtubules). – Removing enzyme that puts on tyrosine causes multiple axons to form. Tubulin Post-translational modifications Tubulins can lose their carboxyterminal tyrosine. This can be replaced by an enzyme tubulin tyrosine ligase (TTl). Kinesins that select for axons Stabilization of axon/dendrite is due to Filter at Axon Hillock • Soon after axon determination, ankyrin G and actin filaments form at axon hillock. • This generates a diffusion barrier between axon and soma. • Only some motor proteins can make it through this barrier, limiting transport down the axon. Selective retention of sodium channels at axonal hillock • Cytoplasmic region between segments II and III is sufficient • Contains two motifs; Both are required for full sorting: – One motif is required for association with ankyrin-G (a cytoskeletal protein highly enriched in axonal hillock). This association leads to trapping of protein (triton-insolubility) – One motif leads to endocytosis from Cytoplasmic loop sufficient for axonal hillock localization Garrido et al, Science. 2003 Jun 27;300(5628):2091-4 Red staining is MAP-2. Axons lack MAP2. While CD4 is normally targeted to the soma, addition of the loop from the sodium channel is sufficient to localize it to the axon hillock, Loop has two separate determinants Fache et al, J. Cell Biol. 166: 571-578 One region (aa1010-1030) is required for endocytosis association, but not for interaction with ankyrin-G. In contrast, a separate region (as1098-1111) is required for association with ankyrin-G but not endocytosis. Green staining is CD4 staining. Red staining is MAP-2. Sorting to Dendrites • Proteins sorted to dendrites have specific sorting signals for vesicles that are targeted to dendrites; similar to basolateral targeting in MDCK cells. • Microtubules are orientated in both directions in dendrites, retrograde motors are good to get to dendrites. • Selective endocytosis from axon would be bad for dendritic targeting as axon is very long. What we learned today • Vectorial sorting and selective retention are keys to polarization of membranes • Many different sorting signals used in cells – Sorting signals can be present either in the cytoplasm or the lumen • Microtubules are important in differentiation of neuronal processes