Specific Aims: hypothesize

Transcription

1 Specific Aims: Defects in intracellular trafficking of membrane proteins (e.g. ion channels, receptors, transporters) can alter their surface expression and modulate their downstream signaling. Membrane proteins on the plasma membrane are internalized and transported from early to late endosomes. At the late endosome, proteins destined for degradation via lysosomal fusion are internalized into the lumen of late endosomes via membrane invagination and vesicle fission. Proteins that are not sorted into the late endosome can either remain on the limiting membrane for incorporation into the lysosomal membrane, or they may bud from the limiting endosomal membrane for recycling to various cellular compartments including the plasma membrane. Growth factor receptor recycling has been implicated in cell proliferation and signaling, however, molecular mechanisms underlying the recycling of membrane proteins from the late endosomal membrane are unknown. Thus, elucidating the machinery required for recycling vesicle budding from the late endosome would unveil details about a key molecular process that impacts large scale signaling. Dynamin, a GTPase involved in vesicle fission events, is essential in vesicle formation, receptormediated endocytosis, and synaptic vesicle recycling. At the plasma membrane, dynamin wraps around the neck of inward budding clathrin-coated pits, constricts, and severs the vesicle upon GTP hydrolysis. Clathrin is a coat protein that is recruited by its associated adaptor proteins and is involved in membrane budding and vesicle formation. Receptor recycling events that occur from early endosomal compartments are clathrinmediated and dynamin-dependent. Additionally dynamin is required for vesicle budding that transports cargo from the late endosome to the trans-golgi network. However, whether growth factor receptor recycling from the late endosome is clathrin-mediated or dynamin-dependent is unknown. Due to compelling literary evidence, I hypothesize that clathrin-mediated and associated adaptor protein-dependent vesicle formation, as well as dynamin-dependent budding are required for EGFR recycling from the late endosome. Our lab established a cell-free assay that recapitulates 1) late endosome maturation and its dependence on cytosolic components, 2) the inward budding of cargo protein into late endosome internal vesicles, and 3) outward budding of recycling vesicles. We have found that cytosol from the yeast Saccharomyces cerevisiae will support inward and outward budding in this assay, providing a system amenable to genetic manipulation of components required for budding events. Additionally, cytosol derived from yeast deletion strains can be complemented with the corresponding mammalian recombinant protein to rescue the effects of the yeast deletion. By coupling yeast genetics with our cell-free assay we obtained preliminary data suggesting that dynamin is required for outward but not inward, budding of epidermal growth factor receptor (EGFR)-containing vesicles from crude endosomal membranes. This proposal will 1) determine whether outward budding from late endosomes is clathrin-mediated and will reveal the adaptor proteins that facilitate budding and 2) determine whether EGFR recycling from the late endosome is dynamin-dependent. Specific Aim 1: Determine whether adaptor and coat proteins act to enable outward budding of EGFRcontaining vesicles from the late endosome. To determine which adaptor proteins are involved in the outward budding of late endosome vesicles I will a) use our cell-free approach coupled with yeast deletions of individual adaptor proteins. I will focus on adaptor proteins that have been implicated in receptor recycling from endosomal compartments (e.g. amphiphysin, Eps15, AP-5). To determine whether the EGFR-vesicle budding from the late endosome is clathrin-mediated, I will b) use yeast cytosol derived from a clathrin knockout strain. If outward vesicle budding is inhibited, yeast deletion strains can be complemented with the corresponding mammalian recombinant protein. Finally, I will c) characterize adaptor and coat protein localization on late endosomal membrane by immuno-gold labeling a purified late endosome population with antibodies corresponding to adaptor and clathrin proteins determined to facilitate outward budding in experiments 1a and 1b. Adaptor and coat protein recruitment to late endosomes will be quantified following ligand stimulation as compared to steady state. Specific Aim 2: Determine whether dynamin is required for outward budding of EGFR-containing vesicles from the late endosome. First, I will a) use late endosomal membranes in the cell-free assay to determine whether each dynamin isoform can rescue inhibited outward vesicle budding in a dynamin deleted background. Next, I will b) use a temperature sensitive mutant of mammalian dynamin, that decreases membrane fission following temperature shift, in combination with flow cytometry to quantify EGFR recycling in the presence and absence of functional dynamin. This experiment will shed light on the role of dynamin in receptor recycling in whole cells. Finally, I will c) quantify dynamin recruitment to late endosomes following ligand stimulation using immunoelectron microscopy. Comparing dynamin recruitment following ligand stimulation compared to steady state will provide evidence of dynamin-dependent EGFR recycling from the late endosome.

2 Significance i. Membrane protein trafficking. Cells respond to their environment and communicate with one another via ligand-induced activation of transmembrane receptors at the plasma membrane. The number of membrane proteins on the cell surface, and the time they spend signaling following their activation, are essential for regulation of cellular homeostasis, plasticity, growth, and differentiation 1. Endocytosis of membrane proteins is critical in tuning their signaling responses by 1) modulating the number of receptors present on the surface and 2) regulating downstream signaling effects of receptor activation 2. Specifically, endocytic trafficking of the cell surface receptor, epidermal growth factor receptor (EGFR), is a key mechanism in the modulation of its downstream signaling 2,3. Upon epidermal growth factor (EGF) binding, EGFR is internalized into transport vesicles via clathrinmediated endocytosis 4 and following uncoating, transport vesicles fuse with early endosomes marked by early endosome antigen 1 (EEA1) and Rab5 5. At the early endosome, EGFR can either bud off to recycle back to the plasma membrane or remain on the limiting membrane of the organelle. Early endosomes and the remaining proteins will either 1) mature into recycling endosomes upon loss of Rab5 and association of Rab11 or 2) mature into late endosomes (LE) upon formation of intraluminal vesicles and association with Rab7 and Lamp1 5,6. EGFR sorted into intraluminal Recycling endosome Extracellular Intracellular iii. Vesicle budding machinery. Both clathrin and dynamin have been associated with mechanisms of vesicle budding and formation in intracellular budding events throughout the cell. Specifically, dynaminmediated clathrin-coated vesicles bud from early and recycling endosomes for transport of cargo from the endosomes to the plasma membrane Dynamin also localizes at LEs and facilitates protein trafficking to the Trans Golgi Network (TGN) 40 and lysosome 41. Although there is evidence suggesting 1) clathrin-mediated, dynamin-dependent vesicle budding in receptor recycling from early and recycling endosomes, and 2) dynamin-mediated vesicle formation from the LE, the functional role that adaptor, clathrin coat, and dynamin play in successful EGFR-vesicle budding from the LE are unknown. Therefore I hypothesize that clathrin- Clathrinmediated endocytosis Early endosome Late endosome vesicles (ILVs) of the LE are subject to degradation upon LE-lysosome fusion 7,8. EGFR that has not been incorporated into ILVs and remains on the limiting membrane of the endosome may either bud from the endosomal membrane for recycling to various cellular compartments, including the plasma membrane 9 19, or will be incorporated into the membrane of the lysosome upon MVB-lysosome fusion. Understanding the trafficking of EGFR is imperative because of the receptor s ability to signal throughout the endocytic pathway 20,21. Thus, recycling from the LE allows for continued EGFR signaling followed by reactivation at the plasma membrane 18,19,22,23 ; however no conclusive mechanisms or machinery involved in EGFR recycling from the LE to the plasma membrane have been defined. ii. Clathrin-mediated endocytosis. Machinery involved in budding events is often redundant in that trafficking of the cargo can be mediated by similar mechanisms whether at the plasma membrane or elsewhere in the cell. Clathrin-mediated endocytosis (CME) is the primary mechanism involved in EGFR internalization at the plasma membrane. CME of EGFR is initiated by binding of its ligand at the plasma membrane resulting in a conformational change, receptor dimerization, and autophosphorylation 4. Ligand-mediated activation of EGFR recruits adaptor proteins (AP-2, Epsin, Eps15, amphiphysin) to the cytoplasmic tails of the receptors to generate membrane curvature and recruit downstream proteins involved in CME 28,29. A pit begins to form in the membrane and the adaptor proteins recruit clathrin that form a lattice around the budding pit and mediate membrane bending and vesicle size Finally, dynamin, a large GTPase, is recruited by adaptor proteins and assembles around the elongated neck of the budding vesicle Upon GTP hydrolysis, dynamin undergoes a conformational change that results in membrane fission and vesicle budding 2,31,36. The elegant coordination in the recruitment of associated adaptor proteins in clathrin-mediated vesicle formation and the subsequent membrane fission by dynamin is critical in budding of EGFR vesicles from the plasma membrane, and is therefore important in mediating its downstream signaling. TGN Lysosome Figure 1. Schematic endocytic pathway. Budding machinery, clathrin and dynamin, are required for receptor recycling from early and recycling endosomes. Whether these proteins are required for recycling from the LE is unknown 66.

3 mediated and associated adaptor protein-dependent vesicle formation, as well as dynamin-dependent budding are required for EGFR-specific recycling from the late endosome. iv. Innovation. This proposal is the first to directly investigate not only localization but also the function of specific cytosolic components in EGFR recycling from the LE by utilizing cell-free, whole-cell, and microscopy approaches. Recent studies have used microscopy to show that dynamin localizes to the limiting membrane of LEs, however these techniques do not possess the capability to show a definitive function of dynamin at the LE 40,41. Our cell-free approach not only allows for the separation of endocytic compartments (early endosome, recycling endosome, and late endosome), but also allows for a quantitative interrogation of the effect specific cytosolic components have on receptor internalization or budding in the endocytic pathway. Elucidating the molecular mechanisms of EGFR trafficking will provide valuable insight into the details of EGFR signaling regulation. Core Methods Cell Free Sorting Assay 42 : HeLa cells will be grown to 80% confluence and serum-starved for 2 hours. Cells will then be stimulated with ligand (EGF) for 20 minutes to allow for the maximum number of receptors to be internalized and reach endosomes. Cells will be drawn through a 30-gauge needle and lysate will be centrifuged to remove debris. The resulting supernatant will be loaded on top of a continuous OptiPrep gradient (Sigma, 10-20%) and centrifuged in a swinging bucket rotor. LEs will be obtained from peak fractions determined by organelle markers (LAMP1 and Rab7) 43. The resulting endosomal pellet will be incorporated into a reaction consisting of a) endosomal membranes, b) cytosol, c) ATP regeneration system, and d) homogenization buffer. The reactions are supported by both mammalian and yeast (Saccharomyces cerevisiae) cytosol. Reactions will then be placed in a 37 C water bath for 3 hours. Incubation of endosomes with ATP and cytosol will result in formation of internal vesicles and outwardly budded recycling vesicles (Fig. 2). Following water bath incubation, reactions will be centrifuged to separate LEs (in pellet) and outwardly budded vesicles (in supernatant) 42. The resulting supernatant is spun for 1 hour to isolate outward budded Serum-starved cells a Overnight gradient endosome separation b Trypsin Cytosol derived from different model systems support the sorting assay, including: + ATP, cytosol 3 hour incubation Trypsin Human cell lines Saccharomyces cerevisiae Drosophila melanogaster Western blot Figure 2. Cell-free reconstitution of LE sorting and recycling. c Centrifuge SUPERNATANT PELLET d Ultracentrifuge vesicles, the resulting pellet will be resuspended in sample buffer and run on Western Blot probing for EGFR, reflecting outward budded vesicles in the sample (Fig. 2d). Simultaneously, the LE pellet will be resuspended in homogenization buffer and trypsin that removes the C-terminal epitope of receptors remaining on the limiting membrane of the endosome. Receptors that have been internalized are protected from trypsin cleavage and are thus detectable via Western Blot (Fig. 2c). Endosomes that have not been incorporated into reactions are either run on a Western Blot probing for EGFR (Fig. 2a) or are incubated with trypsin (Fig. 2b) reflective of initial amount of receptor on the limiting membrane and the initial amount of receptor internalized, respectively. All cell-free experimental data will be analyzed using one-way analysis of variance (ANOVA) and the appropriate post-hoc test. Immuno-electron Microscopy: HeLa cells will be serum-starved for 2 hours and incubated with ligand (EGF) on ice for 1 hour. Media will be aspirated, cells will be washed of unbound ligand and incubated in pre-warmed media for time points between 10 and 100 minutes (10 minute increments). Following incubation, cells will be lysed and loaded onto an OptiPrep gradient as described above. LEs isolated from an OptiPrep gradient will be loaded and fixed (4% PFA) onto glowdischarged carbon-coated copper grids. Grids will then be blocked, incubated in primary antibody and washed before incubation in gold-conjugated secondary antibodies. Grids will then be negatively stained for structural identification of organelles. Endosomes will be randomly selected from the fraction by an experimenter blind to the treatment groups. Images of isolated endosomes will be taken on a JEOL1400 electron microscope for analyses. A power analysis based on preliminary results will determine sample size for number of observations and experimental groups. Quantitative data will be analyzed using one-way ANOVA and the appropriate post-hoc test.

4 Temperature Sensitive Flow Cytometry: This method will use a stable HeLa cell line (tta-hela) expressing a chimeric tetracycline transcription activator 44. tta-hela cells contain dynamin 2 with a site-directed mutation in the glycine at position 273 to aspartic acid 44. This point mutation enables a tetracycline and temperature dependent dynamin defect in GTP binding and hydrolysis 44, hours prior to the experimentation, tta-hela cells will be cultured in the presence (induced) or absence (uninduced) of tetracycline. Cells will then be serum-starved and stimulated with EGF ligand. Following 20-minute stimulation allowing for most internalized receptors to reach the endosomes, cells will either be incubated in the permissive temperature (32 C) or nonpermissive temperature (38 C). Cells being shifted to the non-permissive temperature will be media aspirated and replaced with pre-warmed 38 C media before incubation. Following time points ranging between 10 and 100 minutes, cells will be lifted using 1X PBS + EDTA, washed, and resuspended in FACS buffer (1X PBS, 2% FBS, 1mM EDTA). Cells will be incubated with AlexaFlour488 anti-human EGFR antibody (BioLegend), fixed for 20 minutes in 2% paraformaldehyde, washed with FACS buffer, filtered through 70um filters, and analyzed on a Cytoflex S using CytExpert software (Beckman Coulter) 19. Mean fluorescence intensity (MFI) will be measured for each sample and data will be analyzed using FlowJo software (FlowJo, LLC). Positive (maximum receptors on the cell surface) and negative (maximum receptors internalized) controls will be used and a cell viability stain will account for cell survival. MFI across experimental groups and time points will be analyzed using a two-way ANOVA and appropriate post-hoc analysis. Approach Specific Aim 1: Determine whether adaptor and coat proteins act to enable outward budding of EGFRcontaining vesicles from the late endosome. Rationale: During vesicle budding events, adaptor proteins are first recruited to membranes to cluster cargo and induce membrane curvature, subsequently coat proteins (e.g. clathrin) are recruited and begin to assemble around the bud recruiting additional adaptors and dynamin 31,46. Evidence suggests that adaptor proteins-1 (AP-1), -3, and -5 are associated with late endosomal membranes and cargo recycling from recycling and LEs 47,48. Epsin, (EGFR pathway substrate 15) Eps15, and Amphiphysin are adaptor proteins that play a role in membrane bending, cargo binding and clathrin recruitment at the plasma membrane 14,31. ACAP- 1 49,50 and GGA3 51 are required for clathrin-dependent recycling of receptors from early endosomal compartments to the plasma membrane. Additionally, clathrin coats are required for recycling of cargo from early and recycling endosomes to the plasma membrane 49,51. However, adaptor and coat proteins required for cargo recycling from the LE have not been identified. Preliminary mass spectrometry analysis of recycling vesicles that bud from a crude endosomal population revealed that AP-1, -3, and clathrin coats are associated with these budded vesicles. These results were obtained from a crude endosomal population and thus conclusions cannot be drawn in regards to proteins specifically involved at the LE 43. This aim will elucidate which adaptor proteins are involved in receptor recycling from the LE, and whether these vesicles form in a clathrin-dependent manner. Based on subcellular localization and their role in recycling vesicle formation, I hypothesize that AP-5, Eps15 and ACAP-1 and clathrin coats are involved in recycling vesicle budding from the LE. Experimental Design: Experiment 1a) Determine adaptor proteins involved in outward vesicle budding from the late endosome. To identify the mechanisms underlying both inward and outward budding from the LE, our lab has established a cell-free assay that allows examination of both inward and outward budding from the same donor membranes (detailed in Core Methods section) 43,52,53. Wild-type yeast cytosol will be used as a positive control. Vps1 is a dynamin-like yeast orthologue and will be used as a negative control, as it inhibits outward EGFRvesicle budding 43. Experimental groups will include yeast cytosol that has been derived from deletion strains that are mammalian adaptor protein orthologues. Immunoreactivity of EGFR will be determined via Western Blot analysis and is reflective of inward and outward budding. Any yeast knockout strains that significantly inhibit EGFR-containing outwardly budded vesicles from the LE will be incorporated into complementation reactions including the corresponding mammalian recombinant protein. Successful rescue of inhibited outward budding will confirm involvement of mammalian adaptor protein in outward EGFR-vesicle budding from the LE. Results from this experiment will determine which adaptor proteins are involved in EGFR-vesicle budding from the LE in a cell-free model. Anticipated Results: Eps15, AP-5 and ACAP-1 have been shown to be involved in EGFR endocytosis at the plasma membrane 25,54 or have been implicated in budding events from endosomes 41,47,48. Thus, I anticipate that reactions containing cytosol devoid of Eps15, AP-5 and ACAP-1 will inhibit outward budding from the LE and complementation with the recombinant protein will rescue observed inhibition.

5 Potential Pitfalls and Alternative Approaches: There are adaptor proteins (e.g. AP-5) that do not have identified yeast orthologues. Alternatively, cytosol can be derived from HeLa cells that have been immunodepleted of the adaptor protein of interest and subsequently incorporated into cell-free reactions. Experiment 1b) Determine whether clathrin is involved in vesicle budding from the LE. This experiment will use the cell-free sorting assay and contain reactions with wild-type yeast cytosol as a positive control, vps1δ cytosol as a negative control, and reactions with cytosol derived from yeast cytosol with a clathrin heavy chain knockout (CHCΔ). If inhibition of outward vesicle budding is observed, reactions with CHCΔ cytosol will be complemented with recombinant mammalian clathrin to rescue any inhibition effect observed. Anticipated Results: Endocytic compartments are dynamic and often require similar machinery for budding events; therefore, I anticipate that CHCΔ cytosol will inhibit outward vesicle budding from the LE, and that complementation with recombinant clathrin will rescue this inhibition effect. Additionally, I expect that across all trials cytosol devoid of adaptor and coat proteins will have no affect on inward budding events due to the fact that they are cytosolic proteins and not known to be present in the lumen of endosomes. Potential Pitfalls and Alternative Approaches: Though implicated in receptor recycling from endosomes, clathrin are not the only coat proteins involved in intracellular trafficking. COPI and COPII proteins are also involved in budding events at endosomes, endoplasmic reticulum, and TGN If CHCΔ cytosol does not inhibit outward budding, cytosol derived from yeast strains devoid of COP protein orthologues can be tested in the cell-free assay to determine whether they play a role in EGFR-containing recycling vesicle formation from the LE. Testing alternative coat proteins would enable the confident conclusion that EGFR recycles from the LE in a coat-dependent manner. Experiment 1c) Characterize late endosome localization of adaptor and coat proteins. Presence of adaptor and coat proteins in late endosomal fractions will be confirmed using immuno-gold labeling and electron microscopy (EM) 39,40. HeLa cells will be starved, stimulated, incubated for time points between minutes, lysed, and put on an overnight OptiPrep gradient for separation of endosomal fractions. Several time points were chosen to capture the maximum number of recycling events that occur at the LE and were chosen based on the amount of time it takes EGFR to be internalized, reach endosomes and be recycled 19,41. Endosome fractions collected from the gradient will be probed for early, late and recycling endosome markers (EEA1, Lamp1, and Rab11) with Western Blot as compared to cell lysate. Intensity of Western Blot bands will indicate peak immunoreactivity fractions containing each population of endosomes 43. Next, the LE peak fraction(s) will be fixed onto EM grids (described in Core Methods) and incubated with commercially available antibodies against its respective marker (Lamp1) and the corresponding secondary antibody conjugated to 12- nm gold 15,59,60. Positive gold labeling associated with endosomes will be counted and quantified to determine the purity of the LE fraction. Endosomal compartments are very dynamic, thus gradient fractions that are determined as peaks of LE marker via Western Blot may have some early or recycling endosome contamination. In this experiment, purity of the fraction takes precedence over endosome yield. Therefore, the fraction selected will be probed for LE marker Lamp1 and the percentage of purity can be calculated by determining what proportion of endosomes on the grid contains the LE marker. Fractions selected can then be optimized until fractions are ~90% pure. This measure would ensure that any adaptor or coat protein probed for in the fraction is indeed present on the LE. Once purity of the fraction is confirmed, fractions will be incubated with commercially available antibodies against the protein of interest (e.g. AP-5, Epsin, clathrin) and subsequently with a gold conjugated secondary antibody. The number of positively gold-labeled endosomes as well as number of gold particles per endosome will be quantified via electron microscopy and compared to HeLa cells that have not been stimulated with EGF. Control conditions include 1) cells that have been serumstarved, but not stimulated with EGF and 2) cells that have neither been serum-starved nor stimulated with ligand. Anticipated Results: I anticipate that the number of endosomes with the protein of interest may not change drastically with or without EGF stimulation due to steady state localization, rather the average number of gold particles per endosome (representing adaptor or coat protein) would increase when cells are stimulated with EGF compared to cells that have not been stimulated. Additionally, when cells are starved, but not stimulated there may be a decrease in adaptor and coat proteins due to increased recruitment to the plasma membrane. Potential Pitfalls and Alternative Approaches: While an increase in adaptor and coat protein levels at LEs following EGF stimulation is convincing evidence that these proteins get recruited to facilitate EGFR recycling, this conclusion cannot be definitive by probing for adaptor and coat proteins alone. Colocalization of adaptor and coat proteins with EGFR can be confirmed via double gold labeling with an antibody against the C-terminal of EGFR and a 5-nm gold conjugated secondary antibody. However, due to the presumed proximity of the two proteins during vesicle budding, there is the potential risk of secondary competition due to the size of the gold

6 particles. This technical pitfall can be addressed by probing for EGFR and adaptor / coat proteins separately. Quantitation of the proportion of LEs enriched in EGFR and could provide confidence in the interpretation that adaptor and coat proteins present at the LE are involved in EGFR-specific budding. Specific Aim 2: Determine whether dynamin is required for outward budding of EGFR-containing vesicles from the late endosome. Rationale: Dynamin plays a key role in membrane fission events by assembling into a helical polymer that constricts upon GTP hydrolysis 17, Dynamin has three mammalian isoforms (dynamin 1, 2, and 3) that are thought to have overlapping yet discrete functions Dynamin 1 is primarily expressed in neuronal tissue and plays a role in rapid synaptic vesicle recycling, dynamin 2 is expressed ubiquitously and facilitates endocytosis of surface receptors and recycling of membrane proteins 61,62, and dynamin 3 is expressed in low amounts in neurons and acts in dendritic spines at the postsynapse 36,41, Dynamin 2 has been implicated in receptor recycling from early and recycling endosomes 61, additionally dynamin 2 has been shown to localize with LEs via electron microscopy and thought to facilitate retrograde transport from the LE to the TGN 40. Furthermore, preliminary Mass Spectrometry results of recycling vesicles from a crude endosome population identified dynamin 2 as an associated vesicle protein 43. There is striking evidence that implicates dynamin 2 at LEs, and dynamin 2-dependent receptor recycling from earlier endosomal compartments. However, whether outward budding from the LE is dynamin-dependent is not known. In this Aim, I will determine whether dynamin facilitates outward budding from the LE and examine the role of dynamin in EGFR recycling from LEs in a whole-cell model. In addition, I will characterize dynamin presence on the membrane of LEs following EGF stimulation using electron microscopy. Due to strong evidence suggesting endogenous dynamin 2 presence at the LE 40,41 and the role dynamin 2 plays in receptor recycling from early endocytic compartments, I hypothesize that recombinant dynamin 2 will rescue outward budding of EGFR-containing vesicles from the LE in a dynamin deleted background. Additionally, the whole cell technique proposed in this aim will demonstrate the functional role dynamin plays in EGFR recycling at the LE under physiologic conditions. Experimental Design: Experiment 2a) Test which dynamin isoform(s) is involved in outward budding from the LE. This experiment will use the cell-free sorting assay described above to test whether each dynamin isoform can rescue outward budding of EGFR-containing vesicles from LEs in a dynamin-deleted background. Each experiment performed will contain reactions with wild type yeast cytosol (positive control), reactions containing vps1δ cytosol (negative control), and reactions containing vps1δ cytosol complemented with recombinant mammalian dynamin 1, 2 or 3 (experimental). Inward and outward budding will be measured via Western Blot as described in the Core Methods section. Anticipated Results: I expect that recombinant dynamin 2 added into a dynamin deleted background will rescue outward budding from the LE due to its ubiquitous cellular expression and presence on LEs 40,41. Dynamin 1 and 3 are not endogenously expressed in HeLa cells, however if they do facilitate outward budding from the LE in a cell-free reaction, this result may reveal an undiscovered role of dynamin 1 and 3 in receptor recycling from LEs in neurons. Finally, I expect that across all trials vps1δ cytosol and the addition of recombinant dynamin will have no affect on inward budding events due to the fact that dynamin is cytosolic and not known to be present in the lumen of endosomes. Potential Pitfalls and Alternative Approaches: It is possible that the effect seen by addition of recombinant dynamin isoforms is not a physiological effect due to spontaneous oligomerization of dynamin around the neck of a budding membrane 51. This potential pitfall will be addressed in Experiment 2b where the role of dynamin in EGFR recycling from the LE will be examined in whole cells. Additionally, since dynamin 1 and 3 are not endogenously expressed in HeLa cells, the Move cells to nonpermissive temperature Serum starve cells Stimulate cells with EGF Quantify cellsurface EGFR expression via flow cytometry Move cells to permissive temperature Figure 3. Temperature sensitive dynamin experiment schematic. rescue of outward budding from the LE observed with recombinant dynamin 1 and 3 could suggest that dynamin plays a role at LEs in cells where they are expressed (neurons). This can be addressed by performing the cell-free assay with LEs isolated from a neuronal cell line (SH-SY5Y) to determine whether dynamin isoforms are required for outward budding from the LE.

7 Experiment 2b) Test whether dynamin inhibits EGFR recycling from the MVB in whole cells. Although the cell-free approach allows for a functional and rapidly quantifiable investigation of dynamin-dependent vesicle formation from the LE, this whole-cell experiment will show whether dynamin is required for EGFR recycling from the LE under physiological conditions. I will use a stable HeLa cell line containing a tetracyclineinducible temperature sensitive mutant of dynamin 2 (tta-hela) 44,45 to specifically perturb dynamin at a point in time when maximal number of internalized receptors are at the LE, as detailed in the Core Methods section. After stimulation with ligand and receptor internalization, I will transfer cells to a permissive (functional dynamin) or non-permissive temperature (non-functional dynamin). Cells will either be kept at their respective temperature for time points ranging from minutes. Following incubation, EGFR recycled to the plasma membrane will be determined via tagging cell surface EGFR with an AlexaFluor488 anti-human EGFR antibody (BioLegend). Mean fluorescence intensity (MFI) will be measured by flow cytometry (Core Methods section). Control conditions include 1) cells that have not been tetracycline induced and are stimulated for 20- minutes, which will give a baseline fluorescent measure following receptor internalization, 2) cells that have not been tetracycline induced and are serum-starved to indicate maximal cell-surface EGFR expression, 3) cells that will not be tetracycline-induced and moved to both permissive and non-permissive temperatures following EGFR internalization, and 4) cells that have been tetracycline induced and moved to the permissive temperature. Anticipated Results: I anticipate that control condition 1) will yield very low levels of MFI and will indicate the relative amount of EGFR that is not internalized following EGF stimulation, 2) will have an extremely high MFI because cells have been serum-starved and high levels of EGFR are expressed on the cell surface, 3) if cells have not been tetracycline induced, dynamin will not undergo the temperature sensitive mutation thus MFI would not change based on the temperature of incubation, which rules out temperature shift as a confounding variable, and 4) cells that have been tetracycline induced will express the temperature sensitive dynamin mutant, however MFI obtained from this group should be comparable to the previous condition (3), which indicates dynamin function is only altered at the non-permissive temperature. Finally, I expect that when tetracycline induced cells are moved to the non-permissive temperature following EGFR internalization, there will be a decreased in MFI (similar to MFI in control condition 1), compared to cells kept at the permissive temperature where dynamin is functional and outward budding from the LE will not be inhibited. These results would confirm that dynamin does play a role in receptor recycling in whole cells and will provide an accurate time course of EGFR recycling 39,61. Potential Pitfalls and Alternative Approaches: Endocytic compartments are dynamic, which may make it difficult to differentiate between recycling events occurring from the recycling endosome versus recycling from the LE if there are no obvious waves of recycling in the time-course. To address this issue, HeLa cells can be transfected with a dominant negative form of Rab11, which will block recycling from Rab11-positive recycling endosomes 65. It is likely that fast recycling from the early endosome would occur before cells are switched to the non-permissive temperature, thus any recycling seen in cells transfected with the Rab11 dominant negative could confidently be interpreted as late endosomal recycling 19. Experiment 2c) Characterize late endosomal localization of dynamin. As described in Experiment 1c, cells that have been starved, stimulated and incubated for time points between 10 and 100 minutes will be lysed and LEs will be collected from an OptiPrep gradient. Purity-optimized LE fractions collected from the gradient will be incubated with commercially available antibodies against dynamin 2 and the corresponding secondary antibody conjugated to 12-nm gold 39,40. Positive-gold labeling for dynamin per structure as well as number of structures with gold labeling will be counted and quantified for each sample to determine differences in dynamin recruitment to late endosomal structures upon EGF stimulation. Cells that have been starved and not stimulated with EGF in addition to cells neither starved nor stimulated will serve as control conditions. Anticipated Results: I expect an increase in the number of gold particles (dynamin 2) per endosome in cells that are stimulated with EGF as compared to cells that have been neither starved nor stimulated (steady state). Additionally, when cells are serum-starved and not stimulated, an abundance of dynamin 2 is recruited to the plasma membrane to facilitate clathrin-mediated endocytosis, thus, in this condition number of gold particles per endosome may be decreased compared to steady state cells that have been neither starved nor stimulated. Potential Pitfalls and Alternative Approaches: As mentioned in Experiment 1c, an increase in dynamin 2 levels at LEs following EGF stimulation is persuasive evidence that dynamin gets recruited to the LE to facilitate EGFR recycling. However to confirm dynamin colocalization with EGFR at LEs, double gold labeling can probe for EGFR and dynamin simultaneously (described in Experiment 1c). This additional experimental condition would provide concrete evidence that dynamin is recruited to EGFR-containing budding vesicles from the LE.

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