Regulating Endocytic Trafficking from the Late Endosome - Assignment Example

FACULTY OF SCIENCE MODULAR DEGREE SCHEME BSc HONOURS DEGREE IN (BIOMEDICAL SCIENCE) Investigating the Regulation of Growth Factor Trafficking Pathways as Novel Approach to Increasing Proliferation of Insulin-Producing Cells with Application to Diabetes Therapy Contents page Abstract ………………………………………………………………………... 3 Introduction ………………………………………………………………........ 4-7 Methods and materials ………………………………................................... 8-17 Immunohistochemistry staining of paraffin-embedded mouse pancreas tissue sections Fluorescence staining of INS-1 cells grown on cover slips to determine expression of Rab7 Fluorescence staining of INS-1 cells grown on cover slips to determine expression of Rab7 Results………………………………………………………………………… 18-21 Discussion …............................................................................................ 22-24 Conclusion ………………………………………………………………….... 25 Reference ……………………………………………………………………. 26-28 Acknowledgments ……………………….………………………………….. 29 Abstract The internal storage pool of insulin is mainly located in the transport vesicles in the subcellular locations. When homeostatic mechanisms prevail, and there is a need for extracellular hormones, these vesicles undergo exocytosis. This process follow a signal mediated trafficking system, and the Rab family of small GTP binding proteins may represent the key components of vesicular trafficking. Consequently depending on the need for specific protein functions, different Rab proteins are synthesized, recruited, and localized in the subcellular vesicles. Rab7 proteins are located in the late endosomes, participating in the endosomic Golgi transport. Rab7 has been detected in many tissues by immunofluorescence method. With the use of immunofluorescence and confocal microscopy, the intracellular distribution of Rab7 can be examined. At the molecular level, Rab7 has been known to control endosomal trafficking via its GTPase activity. It has been suggested that active GTP-bound Rab7 increases the velocity of dynein-mediated endosomal transport along the microtubules. Several diverse sets of physiological mechanisms of the cell have been demonstrated to be Rab7 mediated. The glucose load in the type 2 diabetics can thus be handled in the manner indicated, and hence this could be a possible signal mediated trafficking mechanism that may be involved in control of extracellular glucose levels in these individuals. In this experiment, localization of Rab7 protein vesicles through a novel method has been demonstrated, and this could serve as a novel method of intervention of the pathway of cellular expressions, so a drug target can be developed to treat these individuals more effectively. Section 1 Introduction The beta-cells in the pancreas secrete primary polypeptide hormone product that is packaged into large membrane-bound secretory vesicles or beta-granules at the trans-face of the Golgi complex for release via the regulated secretory pathway (Feng and Arvan, 2003, 31486-31494). Most beta-granules reside in an internal storage pool, but in response to a physiological stimulus such as elevated blood glucose, a sub-population of these granules are transported to plasma membrane and undergo exocytosis, resulting in the extracellular secretion of insulin (Kalina et al., 1989, 19-23). The Rab family of small GTP binding proteins represents key components of vesicular trafficking, mediating a range of diverse functions, such as, vesicle formation, vesicle transport, organelle motility, vesicle docking and fusion, and exocytosis and endocytosis. Different Rab proteins can participate in these processes depending on the specific functions of the protein involved and its unique subcellular localization. For example, Rab7 is located in late endosomes, participating in late endosome Golgi transport (Jordens et al., 2005, 1070-1077). (Photomicrograph of a Cell Showing Green Rab7 Proteins from www.cellsignal.com/products/2094.html) accessed on 2009-04-24 . Like any other endocrine cell, the pancreatic beta-cells are also continually regulating the balance between hormone biosynthesis, secretion, and intracellular degradation in order to maintain the optimal cellular hormone levels (Miettinen et al., 2008, 280-285). In pancreatic beta-cells, insulin stores in the cells; more specifically, beta-granules are primarily maintained by efficient up-regulation of the biosynthesis of pro-insulin at the level of translation. This mechanism rapidly replenishes the used-up insulin through the process of exocytosis. In a normal healthy individual, degradation of insulin within the beta-cells plays minor role via crinophagy. However, this mechanism is not sufficient to maintain insulin storage levels in the optimum range in the diabetics where secretion of insulin in response to diet is dysfunctional (Flatt and Green, 2006, 774-778). Growth hormone (GH) and insulin-like growth factor (IGF)-1 have demonstrable interactions in diabetes. There is evident growth failure associated with poorly controlled diabetes, especially during childhood. It has been demonstrated that total IGF-1 levels are diminished in adolescents with poorly controlled diabetes, and they have often elevated plasma GH levels (Fernández et al., 2001, 1926). One of the major actions of insulin is glucose transport in the insulin target tissues, muscle and fat. This process involves translocation of the insulin-responsive glucose transporter GLUT4 to the plasma membrane. It has been noted that GLUT4 is protein in nature, and they are contained in the intracellular vesicles. These intranuclear vesicles are predominantly localized to a perinuclear compartment in the unstimulated basal state (Minokoshi, Kahn, and Kahn, 2003, 33609-33612). With food challenges and following subsequent insulin stimulation, these vesicles are translocated to the plasma membrane. This process includes multiple steps involving release of vesicles from storage pools, transport to the plasma membrane, proper docking, and fusion with the membrane. It has been postulated that these events are regulated by multiple insulin signalling components (Huang et al., 2002, 2090-2098). Research has demonstrated that different Rab proteins are present in these vesicles utilized for such trafficking in response to a glucose load initiated in these cells. Moreover, these GLUT4 vesicles has been demonstrated to contain a number of associated molecules, such as Rab4, Rab5, Rab7, Rab11, insulin-responsive amino peptidase, and transferrin receptors (Kaddai, Le Marchand-Brustel, and Cormont, 2008, 75-88). The small molecular weight Rab7 has been known to be a regulator of the distal stages of these endocytotic pathways. Data support a role of Rab7 at the early endosome/late endosome interface as well as regulating the transitions from late endosome to lysosome (Ceresa and Bahr, 2006, 1099-1106). Studies have indicated that Rab7 is the regulator of the trafficking of EGF-EGFR complex. It is also known that beta-cell growth and differentiation is mediated by growth factors (Huotari et al., 1998, 1494-1499). Other studies have shown also that endosomal GTPase Rab7 controls the endosomal trafficking (Saxena et al., 2005, 10930-10940). Thus it can be hypothesised that controlling beta-cell proliferation through modification of Rab7 which can modify the growth factor mediated beta cell proliferation, which can enhance insulin secretion in diabetic patients. Therefore, modification of the cellular signalling system through manipulation of Rab7 can serve as a unique pathway for diabetic treatment. Other studies have revealed the presence of Rab proteins on GLUT4 vesicles which may play important roles in insulin-regulated glucose transport both in health and disease (Ishikura, Koshkina, and Klip, 2008, 61-74). Rab7 has been found to be non-speciafically associated with GLUT4 vesicles in mass spectroscopy analysis. Intracellular localisation of Rabs has been done in other studies using confocal immunofluorescence microscopy (Roach et al., 2007, 353-358). It has been demonstrated that endogenous Rab proteins are overexpressed in diabetes type 1, and overexpression of Rab proteins is known to impair the endogenous trafficking pathways (Segev, 2001, re 11). Therefore, an experiment to locate these proteins can serve as both a diagnostic measure as well as a method to target therapy. In this experiment, an investigation on regulation of the growth factor trafficking pathways that can regulate increasing proliferation of beta granules on the basis of Rab7 protein expression will be done in order to find out whether it can serve as a novel mechanism targeted to diabetes type 1 therapy. Due to the reasons elucidated above, it can be an idea worthwhile to determine the sub-cellular localization of Rab 7 protein by immunohistochemistry, which may allow us to follow the regulation of Rab 7 gene expression by the inflammatory cytokines in the INS-1 beta-cell lines; if it is a beta-cell, it will demonstrate insulin granules within the cellular cytoplasm. In that scenario, the movement of Rab7 proteins within the cell starting from the cell membrane would indicate a cellular trafficking pathway associated with insulin response. In this experiment, this will be demonstrated through cellular preparation, immunohistochemistry staining and examination under a confocal microscope. Key Words: Beta cells, Crinophagy; Cellular trafficking pathways; Endocytosis; Exocytosis; GLUT4 vesicles Insulin-like growth factor (IGF)-1; Lysotracker and Lysosensor Probes; Rab family Section 2 Materials and Methods Materials A. Solutions and Reagents 1. 10 X Phosphate Buffered Saline (PBS) To prepare 1 L each tablet dissolved in to 100 mL; 10 PBS tablets were dissolved in 1000 mL of distilled water. 2. Citrate Buffer pH 6.0 To prepare 1000 mL Sodium hydroxide (NaOH) Tween 20------------------0.5 mL Citric Acid----------------1.92 g Sodium Citrate----------2.94 g Distilled Water----------1000 mL pH was adjusted to 6.0 The mixture was boiled and mixed well with Steria machine. Since the process is hazardous, this was done in a fume cabinet. The histochemical diagnosis of the subcellular protein components depend on successful staining of paraffin sections. Selection of antigen retrieval techniques is crucial for successful staining of paraffin sections. Different antibodies may require different antigen retrieval techniques, and these need to be tested prior to application in actual projects. 3. 3% Hydrogen peroxide, to prepare 100 mL 30% H2O2---------------------10 mL PBS or methanol ------------90 mL 4. Blocking Solution Horse Serum----------------150 µL PBS-----------------------------10 mL These two are mixed well. 5. Primary Antibody Dilution of primary antibody is critical for success in staining. Titration is necessary prior to application in actual projects Anti INS-1---------- made in guinea pig Anti Rab7----------made in rabbit 6. Secondary Antibody An appropriate secondary biotinylated antibody was selected; a titration was done prior to application Anti guinea pig –------- biotinylated Anti rabbit –------------- biotinylated 7. ABC Reagent Depending on sensitivity and morphological requirements, there are varieties of ABC kits and reagents that can be selected from. The most commonly used one is VECTASTAIN ABC Kit from Vector Laboratories. PBS----------------5 mL Reagent A-------1 drop Reagent B-------1 drop These were mixed well and left for 30 minutes in RT before use. 8. Diaminobenzidine (DAB) Reagent: DAB diluent ------------------1 mL Impact DAB-------------------1 drop These were mixed well and were used in the solution immediately. Methods: Paraffin section (DAB): The slides were marked carefully with dates and initials, and following marking, these were placed on the metal holder. These slides were placed in xylene twice for 5 min in fume cabinet, so the tissue sections deparaffinize. The tissue sections to be examined were hydrated with 100% ethanol for twice for 5 min, followed by hydration with 90% ethanol for 3 min, followed by 70% ethanol for 3 min. During this process, adequate precautions were taken so the slides were not dried. After this, the slides were rinsed in distilled water very carefully and were washed well with PBS for 5 min. Once this was done, these slides were boiled in the citrate buffer solution at pH 6.0 for 10 min. These slides were carefully dried by using Kim Wipe, and a pap-pen was used to draw circle around the section. These sections then were blocked using dilute block solution for 30 min at room temperature. The serum was carefully tapped off taking care not to rinse or wipe. Primary antibodies were added for 60 min at room temperature, careful not to allow drying. These preparations were washed in PBS for 2 min and then 5 min. Following this secondary antibodies were added to these preparations, and a 60-min of contact time was allowed, again carefully preventing dryness while in the room temperature. A repeat PBS wash was done for 2 min and 5 min. These were incubated in peroxidase-blocking solution for 15 min, and a repeat PBS wash was done for 2 min and 5 min. ABC was added onto the sections for 30 min at room temperature followed by a repeat PBS wash in the same manner for the same duration. Then DAB was added and allowed to stay twice for 10 min until the development of the stain. Once the stain developed, it was washed in distilled water for 3 x 2 min following which it was stained in hematoxylin for 3 min. The preparation was then washed in running tap water until the stain was washed off, and these were placed in sodium bicarbonate for 2 min to make hematoxylin blue. The slides were then retrieved and washed in distilled water for 5 x 2 min. Dehydration was undertaken in 70% and 90% alcohol for 5 min each, and to ensure complete dehydration, these were placed in 100% ethanol for 5 min twice. These dehydrated preparations were then placed in xylene for 5 min twice in the fume cabinet. On each of these processed sections, a drop of DePex was added, and cover slip was mounted ensuring that there were no bubbles. This was done by tapping slowly on the slide before it dried out. Slides were dried in fume hood. Tissue culture protocol for INS-1 cells and derived lines: Materials 70% ethanol Water bath Growth medium Phosphate buffered saline (PBS) Trypsin - EDTA 75 cm2 flasks T-75 tissue culture flask filled with 30-40 mL Methods This procedure must be done with the researcher wearing gloves with the use of fume cabinet and spraying 70% ethanol. The flask was emptied of media. PBS was added to wash the cells and the flasks and these were rinsed out in a waste beaker. About 2 to 3 mL of trypsin was added. The flask was placed in the incubator for a minute. Four new flasks were labelled with date and initial, and 10 ml of the media were added to each of these flasks. In order to do this, the flask was taken out of the incubator and 10 ml of the media was added and mixed well. Adequate amount of cell suspension was added to these new flasks, and these new flasks were placed in the incubator again, with changes done in every 3 days. Preparation of Slides Method The cultured cells were grown on sterile glass cover slips or slides overnight at a temperature of 37 degrees Centigrade with 1.5 cover slips being placed in 6 multi-well plates. These cover slips were soaked in 70% ethanol in order to make them sterile. These cover slips were kept alone for 30 minutes to allow the ethanol to evaporate. The cells were then carefully added on the cover slips making sure that it covers all the surfaces. These preparations were placed in the incubator overnight at 37 degrees Centigrade which allowed the cells to grow on the cover slip. This was washed briefly with PBS Fixation of Cells Method The preparations were washed briefly with PBS twice and then fixed with cold acetone for 30 min. This was washed with PBS, and glycine buffer was added for 5 min. A repeat wash with PBS was done followed by blocking with dilution serum for 30 min. Primary antibody for Rab was added and allowed to act for 60 min. This was again washed in PBS. Primary antibodies for INS-1 were added for 60 min followed by a wash in PBS. This was followed by addition of secondary antibodies for Rab for 30 min, a PBS wash, addition of secondary antibodies for INS-1, and a final PBS wash. These preparations were mounted with DAPI by adding a drop on the slide, and then the cover slip was placed slowly over it. The sides and the edges were then sealed with a nail polish, and the preparation was then stored in dark at 4 degree Centigrade for a short time. Lysotracker and Lysosensor Probes The LysoTracker probes that consist of a flurophore linked to a weak base that is partially protonated at neutral pH. They permeate through the cell membranes and are concentrated in spherical organelles. For this experiment LysoTracker and LysoSensor probes were designed in the following manner. 1. The 1 mM probe stock solution was diluted to the final working concentration in the growth medium or in the buffer of choice. For the LysoTracker probes, a working concentration of 50 to 75 nM was desired. To reduce potential artifacts, the concentration of the dye was kept as low as possible. 2. For adherent cells, the cells were grown on Cover slips inside a petridish filled with appropriate culture medium. When cells have reached the desired confluence, the medium was removed from the dish, and the pre-warmed (37 degrees Centigrade) probe containing medium was added to it. The cells were incubated for 30 minutes to 2 hours under growth conditions suitable for the growth of a particular cell type. These probe media were then used on slides with Cover slip with 5 different things on it. Cover slip 1: LysoTracker incubated for 30 min and Rab7 primary for 1 hour and secondary antibody for 30 minutes and DAPI Cover slip 2: LysoTracker only incubated for 30 min and DAPI Cover slip 3: Rab7 primary incubated for 1 hour and secondary for 30 min and DAPI Cover slip 4: LysoTraker incubated for 30 min and Rab7 primary antibody incubated for one hour and DAPI Cover slip 5: LysoTracker incubated for 30 min and Rab7 secondary antibody for 30 min and DAPI. Nail polish was used to seal the Cover slips on the slides. Discussion: Rab proteins are a family of small proteins that bind to guanine nucleotides. These regulate vesicular budding and fusion reactions, and thus they are involved in endocytotic trafficking. Rab7 is another variant of this class of proteins that are localized in late endosomes. There is a defined role for rab7 in regulating the later stages of the endocytotic pathway (Ceresa and Bahr, 2006, 1099-1106). Immunofluorescent staining has been able to detect Rab7 protein in the pancreatic cells, and using Western blotting, these proteins were confirmed to be endogenously present in these cells (Saxena et al., 2005, 10930-10940). By immunofluorescence and confocal microscopy, the intracellular distribution of these Rab7 proteins was also confirmed. This shows Rab7 has initially been localized in the cell membrane of the beta cells where insulin had been also demonstrated to be present. However with passage of time as demonstrated in Figure I, the concentration of Rab7 has reduced in the cell membrane by very light green in comparison to figure IIIA. In contrast to this, there are some little dots in the nucleus, indicating vesicular transport leading to fusion of vesicles into the nucleus of these cells. Concomitantly, there is also a reduction in the intensity of insulin in the cytoplasm of these cells. In figures IIIB, similar insulin concentration remains, but there has been an absence of Rab7 on the cell membranes despite persistence of these in the nucleus.. Thus these are Rab7 proteins mainly located in the nucleus of the cells, indicating a cellular trafficking present within the beta cells. Therefore, this experiment indicates a Rab7 mediated cellular trafficking pathway present in the beta cells of the pancreas. Previous work has indicated that Rab7 forms a complex with the molecular motor dynein and dynactin. If this Rab7 is specific, then it would react positively with the Rab7 antibodies used for the precipitation of endogenous Rab7 (Saxena et al., 2005, 10930–10940) It has been shown by this experiment that the Rab7 proteins are regulating a cellular trafficking pathway mediated by the insulin signals where vesicular transport is occurring from the cell membranes areas to the cellular nuclei. This could mean a signal mediated trafficking system. If the Rab7 proteins are located in the signaling endosomes, the general principles of RabGTPase regulation can be applied here to explain this. In general RabGTPases are regulated by guanine nucleotide exchange factors and GTPase activating factors, and some Rab7 interacting proteins have been isolated that might be involved in upstream or downstream signal transduction. Perhaps through this pathway, the insulin regulation is achieved through generation of different proteins that may bind to Rab7GTPase leading to sustenance of Rab7 activity (Jordens et al., 2001, 1680-1685). Results Pancreatic islet is stained with hematoxylin. It stains the nucleus blue and ABC can help identify the nature of the cells present in the islets of Langerhans. On the pancreas section antibody to insulin has been employed to identify the beta cells. Figure 1(a) shows pancreas section stained with no primary antibody; Figure1 (b) shows pancreas section stained with Insulin primary Abs in 1/50 dilution serum; Figure 1(c) shows Insulin primary Abs in 1/100 dilution serum; Figure (d) shows Insulin primary Abs in 1/200 dilution serum; Figure (e) shows Insulin primary Abs in 1/400 dilution serum; Figure (f): Insulin primary Abs in 1/800 dilution serum. It was concluded that 1/400 dilution serum of Insulin primary Abs is employed to identify the natural of the cells (only beta cells) present in the islelt of the Langerhans. In the immunofluorescence staining, the dark green Rab7 was located in the cell membrane, and the nucleus was stained with DAPI. In these staining method two layers of antibodies, primary antibody rabbit anti-Rab7 and secondary antibody FITC were used. In figure I both primary and secondary antibody of Rab 7 is used. Rab 7 is located in the cell membrane as green colour. In figure III (A) both primary and secondary antibody of insulin is used. Insulin represented by red colour in the cytoplasm. In figure III (B), Rab7 could be located in the cell membrane as green colour, and insulin is seen in the cytoplasm as red colour, and the nucleus of these cells appear stained with DAPI. In figures III both the primary and secondary antibodies of both Rab7 and insulin were used. The insulin represented by red colour in the cytoplasm, but the membrane Rab7 appeared very light green, whereas there were few green dots in the nuclei of these cells indicating Rab7 trafficking pathway. This was confirmed in the next slide figures II (A and B) where both primary and secondary antibodies of insulin and only secondary antibody of Rab7 had been used. This indicates that cell membrane Rab7 is absent, whereas, there is nuclear Rab7 present. This indicates a cellular trafficking pathway involving Rab7 proteins associated with insulin secretion in the beta cells of pancreas. Conclusion The aim of the project was to examine the sub-cellular localization of Rab 7 protein by immunohistochemistry. in the INS-1 beta-cell lines. In this experiment as has been demonstrated in the previous section, the Rab 7 was localised in the cell membrane which is in agreement with the hypothesis of this project. When it was later localised in the nuclear membrane, this also indicates vesicle formation and vesicle movement along the endoplasmic actin and tubulin networks and ultimately membrane fusion. This trafficking route suggests a signalling pathway where cell surface proteins are trafficked from the Golgi complex to the cell membrane and recycled. This was demonstrated through cellular preparation, immunohistochemistry staining and examination under a confocal microscope that from the initial location, the Rab7 proteins are detected within the vesicles beginning from the cell membrane through the endoplasmic reticulum to be fused to the nuclear membrane as time passes. This would indicate a cellular trafficking pathway associated with insulin response. There has been substantial evidence that Rab7 proteins regulate major steps in vesicle transport. These steps include targeting of the vesicle to the acceptor membrane, docking of the vesicle, and fusion of the vesicle to the membrane accepting it. This experiment demonstrates translocation of Rab7 proteins from the cell membrane to the nuclear membrane. Thus if IGF is related to insulin secretion, it can be concluded that, through these signalling pathways, the ligand stimulation will also cause IGF to internalise and be transported through the endocytotic pathway. The endocytotic pathway in turn is regulated by Rab7 protein and has been shown to regulate the rate of IGF degradation and recycling as well as signalling mediated by the IGF. References Ceresa, BP and Bahr, SJ. (2006). rab7 Activity Affects Epidermal Growth Factor:Epidermal Growth Factor Receptor Degradation by Regulating Endocytic Trafficking from the Late Endosome. The Journal of Biological Chemistry. 281(2). 1099-1106. Feng, L. and Arvan, P., (2003). The Trafficking of 1-Antitrypsin, a Post-Golgi Secretory Pathway Marker, in INS-1 Pancreatic Beta Cells. J. Biol. Chem.; 278: 31486 - 31494. Fernández, AM., Kim, JK., Yakar, S., Dupont, J., Hernandez-Sanchez, C., Castle, AL., Filmore, J., Shulman, GI., and Roith, DL., (2001). Functional inactivation of the IGF-I and insulin receptors in skeletal muscle causes type 2 diabetes. Genes & Dev.; 15: 1926. Flatt, PR and Green, BD., (2006). Nutrient regulation of pancreatic beta-cell function in diabetes: problems and potential solutions. Biochem Soc Trans; 34(Pt 5): 774-8. Huang, C., Somwar, R., Patel, N., Niu, W., Török, D., and Klip, A., (2002). Sustained Exposure of L6 Myotubes to High Glucose and Insulin Decreases Insulin-Stimulated GLUT4 Translocation but Upregulates GLUT4 Activity. Diabetes; 51: 2090 - 2098. Huotari, M., Palgi, J., And Otonkoski, T., (1998). Growth Factor-Mediated Proliferation and Differentiation of Insulin-Producing INS-1 and RINm5F Cells: Identification of Betacellulin as a Novel b-Cell Mitogen. Endocrinology: 139(4); 1494-1499 Ishikura, S., Koshkina, A., and Klip, A., (2008). Small G proteins in insulin action: Rab and Rho families at the crossroads of signal transduction and GLUT4 vesicle traffic. Acta Physiol (Oxf); 192(1): 61-74. Jordens I, Fernandez-Borja M, Marsman M, Dusseljee S, Janssen L, Calafat J, Janssen H, Wubbolts R, Neefjes J (2001) The Rab7 effector protein RILP controls lysosomal transport by inducing the recruitment of dyneindynactin motors. Curr Biol 11:1680 –1685. Jordens, I., Marsman, M., Kuijl, C., and Neefjes, J., (2005). Rab proteins, connecting transport and vesicle fusion. Traffic; 6(12): 1070-7. Kaddai, V., Le Marchand-Brustel, Y., and Cormont, M., (2008). Rab proteins in endocytosis and Glut4 trafficking. Acta Physiol (Oxf); 192(1): 75-88. Kalina, M., Grimelius, L., Cedermark, B., and Hammel, I., (1989). Insulin and C-peptide co-localization in the beta granules of normal human pancreas and insulinomas. A quantitative immunocytochemical approach. Virchows Arch A Pathol Anat Histopathol; 416(1): 19-23. Miettinen, P., Ormio, P., Hakonen, E., Banerjee, M., and Otonkoski, T., (2008). EGF receptor in pancreatic beta-cell mass regulation. Biochem Soc Trans; 36(Pt 3): 280-5. Minokoshi, Y., Kahn, CR., and Kahn, BB., (2003). Tissue-specific Ablation of the GLUT4 Glucose Transporter or the Insulin Receptor Challenges Assumptions about Insulin Action and Glucose Homeostasis. J. Biol. Chem.; 278: 33609 - 33612. Roach, WG., Chavez, JA., Miinea, CP., and Lienhard, GE., (2007). Substrate specificity and effect on GLUT4 translocation of the Rab GTPase-activating protein Tbc1d1. Biochem J; 403(2): 353-8. Saxena,S., Bucci,C., Weis, J. and Kruttgen, A., (2005). The Small GTPase Rab7 Controls the Endosomal Trafficking and Neuritogenic Signaling of the Nerve Growth Factor Receptor TrkA. The Journal of Neuroscience; 25(47):10930 –10940 Segev, N., (2001). Ypt/Rab GTPases: Regulators of Protein Trafficking. Sci. STKE; 2001: re11. Acknowledgments I would like to thanks, my family for giving me the opportunity to study in Kingston University. I would like to thanks my supervisor Dr. Natasha Hill for giving me the opportunity to participate in this amazing project and for all her guidance, patience and understanding. I would like to thanks Johan Peacock for helping me with taking immunofluorescence pictures. I would like to give my appreciation to member of technical staff in science laboratory in Kingston university especially Steven who assisted me through the project experiment. Lastly I would like to give appreciation to member of technical staff in Kingston University for providing me with the equipment and chemicals: Lindsey Cheeseman, Jayne Reeves, Michael Tsang, Gurmeet Sappal. Read More