- Email: [email protected]
TIRFing out Studies on Glut4 Trafficking
Developmental Cell
Previews phosphorylation of FLN-A downstream of MEKK4. Despite the presence of a large body of work on MAP kinases, little is known about whether MAP kinase signaling directly regulates the actin cytoskeleton or signaling molecules such as small GTPases or PAK that lead to cytoskeleton remodeling. However, MAP kinase is also involved in cell-substratum adhesion, presumably by regulating the assembly of focal complex proteins such as integrin, vinculin, and paxillin (Webb et al., 2004). Cell migration is a highly orchestrated process involving integrated membrane protrusion and membrane attachment to the substratum at the front of the cell and membrane retraction at the rear of the cell. In this respect, the distribution of the extracellular matrix (ECM) protein laminin was discontinuous in the brain of MEKK4 / mice (Sarkisian et al., 2006). Laminin is an integrin substrate and thus an
irregular ECM might affect cell migration. The involvement of stress-associated MAP triple kinase in neuronal migration will shed light on a new layer of the mechanism of cell migration that may occur through the direct regulation of the actin cytoskeleton and/or through the indirect regulation of cellsubstratum adhesion. REFERENCES Chi, H., Sarkisian, M.R., Rakic, P., and Flavell, R.A. (2005). Proc. Natl. Acad. Sci. USA 102, 3846–3851. Fox, J.W., Lamperti, E.D., Eksioglu, Y.Z., Hong, S.E., Feng, Y., Graham, D.A., Scheffer, I.E., Dobyns, W.B., Hirsch, B.A., Radtke, R.A., et al. (1998). Neuron 21, 1315–1325. Nagano, T., Yoneda, T., Hatanaka, Y., Kubota, C., Murakami, F., and Sato, M. (2002). Nat. Cell Biol. 4, 495–501. Robertson, S.P., Twigg, S.R., SutherlandSmith, A.J., Biancalana, V., Gorlin, R.J., Horn, D., Kenwrick, S.J., Kim, C.A., Morava, E., New-
bury-Ecob, R., et al. (2003). Nat. Genet. 33, 487–491. Sarkisian, M.R., Bartley, C.M., Chi, H., Nakamura, F., Hashimoto-Torii, K., Torii, M., Flavell, R.A., and Rakic, P. (2006). Neuron 52, 789–801. Sheen, V.L., Ganesh, V.S., Topcu, M., Sebire, G., Bodell, A., Hill, R.S., Grant, P.E., Shugart, Y.Y., Imitola, J., Khoury, S.J., et al. (2004). Nat. Genet. 36, 69–76. Stossel, T.P., Condeelis, J., Cooley, L., Hartwig, J.H., Noegel, A., Schleicher, M., and Shapiro, S.S. (2001). Nat. Rev. Mol. Cell Biol. 2, 138–145. Takekawa, M., and Saito, H. (1998). Cell 95, 521–530. Tissir, F., and Goffinet, A.M. (2003). Nat. Rev. Neurosci. 4, 496–505. Vadlamudi, R.K., Li, F., Adam, L., Nguyen, D., Ohta, Y., Stossel, T.P., and Kumar, R. (2002). Nat. Cell Biol. 4, 681–690. Webb, D.J., Donais, K., Whitmore, L.A., Thomas, S.M., Turner, C.E., Parsons, J.T., and Horwitz, A.F. (2004). Nat. Cell Biol. 6, 154–161.
TIRFing out Studies on Glut4 Trafficking Xiao-Wei Chen1 and Alan R. Saltiel1,* 1
Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA *Correspondence: [email protected] DOI 10.1016/j.devcel.2006.12.008
Docking and fusion of Glut4 vesicles with the plasma membrane are essential but poorly understood steps during insulin-stimulated glucose transport. Recent studies utilizing TIRF microscopy shed light on these processes and map the sites of possible intervention by insulin from just underneath the plasma membrane.
Insulin stimulates glucose uptake into muscle and adipose cells by promoting the relocation of the glucose transporter Glut4 to the cell surface (Bryant et al., 2002). Despite the general notion that deficiencies in this process represent a primary lesion in the development of type 2 diabetes and related metabolic disorders, little is known about the molecular defects that underlie this pathology (Saltiel and Kahn, 2001). This is largely due to our limited knowledge of precisely how
insulin signaling influences the trafficking itinerary of Glut4. In the basal state, Glut4 is retained in intracellular vesicles and then traffics to the plasma membrane upon insulin stimulation. This process involves multiple steps governed by insulin, including endocytosis into endosomes, sorting of Glut4 into specialized storage vesicles, their retention inside cells, transport along cytoskeletal tracks, tethering, docking, priming, and the final fusion with the plasma
4 Developmental Cell 12, January 2007 ª2007 Elsevier Inc.
membrane. However, the regulated steps in this complex process that are rate limiting, let alone the precise identity of the molecular targets that connect insulin signaling to cellular vesicle trafficking machineries, have yet to be defined. Bai et al. (2007) have investigated the terminal stages of Glut4 trafficking by examining the motion of eGFP-tagged Glut4 molecules using time-lapsed total internal reflection fluorescence (TIRF) microscopy. This approach
Developmental Cell
Previews allows for the selective capture of the docking and fusion events that take place adjacent to the plasma membrane. They observed what appears to be a common pattern for Glut4 vesicle motion during the final stages of its journey to the plasma membrane. Some vesicles, referred to as docked vesicles, seem to remain static within a short range near the plasma membrane for a period (dwell time). Others appeared highly mobile and were thus defined as a part of the predocking stage. Finally, they detected the irreversible movements of some Glut4 vesicles toward the plasma membrane, and they considered these fusing vesicles. Intriguingly, insulin treatment of cells substantially accelerated the fusion rate (8-fold) of Glut4 vesicles with the plasma membrane, but led to only a moderate increase (2 fold) in their docking rate. These data were interpreted to indicate that fusion of Glut4 represented the major site at which insulin acts. In addition, the Akt substrate AS160 (Watson and Pessin, 2006) appears not to participate in this process. Despite some differences with two other recent studies (Gonzalez and McGraw, 2006; Lizunov et al., 2005), the current report is in agreement with the hypothesis that the terminal stages play a pivotal role in regulating Glut4 trafficking. Does this kind of analysis help in our understanding of the steps crucial to insulin action and how these events might be attenuated in insulin resistance? The focus on the final events in Glut4 docking and fusion is a characteristic of TIRF microscopy and allows for a careful examination in real time of
how the transporter finally achieves exposure to the extracellular space. Unfortunately, this approach does not permit assessment of the endocytosis and sorting of Glut4, nor its trafficking from perinuclear regions in the cell, all events that are regulated by insulin and thus potential defects in insulinresistant states. On the other hand, these analyses may now allow investigators to consider many more questions about the process. For example, how are the final stages connected to the penultimate steps in Glut4 vesicle trafficking? Although these earlier steps might not be as responsive to insulin, they may be even more important for building up a reservoir pool of Glut4 vesicles, as studies in other trafficking processes have indicated (Sudhof, 2004). How are Glut4 vesicles selectively presented to the fusion sites, since the SNARE and accessory proteins may not be sufficient to provide this specificity? Vesicle tethering and docking have been suggested to ensure fidelity of trafficking processes and perhaps directly facilitate the fusion events (Munson and Novick, 2006). In the case of insulin-stimulated glucose transport, these events might be carried out by the exocyst complex (Inoue et al., 2003). Hence, the exact links that escort Glut4 vesicles between docking and fusion have to be established. Perhaps the most important question centers on the molecular targets of insulin signaling that choreograph the entire program of Glut4 trafficking. Candidates emerging from recent studies include Rab proteins, SNARE proteins, the exocyst complex, and the
myosin motor Myo1c. Each of these appear to respond to a variety of different signaling events from the insulin receptor, such as phosphorylation, activation of guanyl nucleotide exchange factors and GTPase-activating proteins, and various polyphosphoinositides (Ramm and James, 2005; Watson et al., 2004). It will be of interest to test whether and how these signaling and structural proteins influence the dynamics of the terminal stages of Glut4 trafficking. The new advances in TIRF microscopy may facilitate our efforts to decipher how insulin orchestrates Glut4 translocation. REFERENCES Bai, L., Wang, Y., Fan, J., Chen, Y., Ji, W., Qu, A., Xu, P., James, D.E., and Xu, T. (2007). Cell Metab. 5, 47–51. Bryant, N.J., Govers, R., and James, D.E. (2002). Nat. Rev. Mol. Cell Biol. 3, 267–277. Gonzalez, E., and McGraw, T.E. (2006). Mol. Biol. Cell 17, 4484–4493. Inoue, M., Chang, L., Hwang, J., Chiang, S.H., and Saltiel, A.R. (2003). Nature 422, 629–633. Lizunov, V.A., Matsumoto, H., Zimmerberg, J., Cushman, S.W., and Frolov, V.A. (2005). J. Cell Biol. 169, 481–489. Munson, M., and Novick, P. (2006). Nat. Struct. Mol. Biol. 13, 577–581. Ramm, G., and James, D.E. (2005). Cell Metab. 2, 150–152. Saltiel, A.R., and Kahn, C.R. (2001). Nature 414, 799–806. Sudhof, T.C. (2004). Annu. Rev. Neurosci. 27, 509–547. Watson, R.T., and Pessin, J.E. (2006). Trends Biochem. Sci. 31, 215–222. Watson, R.T., Kanzaki, M., and Pessin, J.E. (2004). Endocr. Rev. 25, 177–204.
Developmental Cell 12, January 2007 ª2007 Elsevier Inc. 5
Previews phosphorylation of FLN-A downstream of MEKK4. Despite the presence of a large body of work on MAP kinases, little is known about whether MAP kinase signaling directly regulates the actin cytoskeleton or signaling molecules such as small GTPases or PAK that lead to cytoskeleton remodeling. However, MAP kinase is also involved in cell-substratum adhesion, presumably by regulating the assembly of focal complex proteins such as integrin, vinculin, and paxillin (Webb et al., 2004). Cell migration is a highly orchestrated process involving integrated membrane protrusion and membrane attachment to the substratum at the front of the cell and membrane retraction at the rear of the cell. In this respect, the distribution of the extracellular matrix (ECM) protein laminin was discontinuous in the brain of MEKK4 / mice (Sarkisian et al., 2006). Laminin is an integrin substrate and thus an
irregular ECM might affect cell migration. The involvement of stress-associated MAP triple kinase in neuronal migration will shed light on a new layer of the mechanism of cell migration that may occur through the direct regulation of the actin cytoskeleton and/or through the indirect regulation of cellsubstratum adhesion. REFERENCES Chi, H., Sarkisian, M.R., Rakic, P., and Flavell, R.A. (2005). Proc. Natl. Acad. Sci. USA 102, 3846–3851. Fox, J.W., Lamperti, E.D., Eksioglu, Y.Z., Hong, S.E., Feng, Y., Graham, D.A., Scheffer, I.E., Dobyns, W.B., Hirsch, B.A., Radtke, R.A., et al. (1998). Neuron 21, 1315–1325. Nagano, T., Yoneda, T., Hatanaka, Y., Kubota, C., Murakami, F., and Sato, M. (2002). Nat. Cell Biol. 4, 495–501. Robertson, S.P., Twigg, S.R., SutherlandSmith, A.J., Biancalana, V., Gorlin, R.J., Horn, D., Kenwrick, S.J., Kim, C.A., Morava, E., New-
bury-Ecob, R., et al. (2003). Nat. Genet. 33, 487–491. Sarkisian, M.R., Bartley, C.M., Chi, H., Nakamura, F., Hashimoto-Torii, K., Torii, M., Flavell, R.A., and Rakic, P. (2006). Neuron 52, 789–801. Sheen, V.L., Ganesh, V.S., Topcu, M., Sebire, G., Bodell, A., Hill, R.S., Grant, P.E., Shugart, Y.Y., Imitola, J., Khoury, S.J., et al. (2004). Nat. Genet. 36, 69–76. Stossel, T.P., Condeelis, J., Cooley, L., Hartwig, J.H., Noegel, A., Schleicher, M., and Shapiro, S.S. (2001). Nat. Rev. Mol. Cell Biol. 2, 138–145. Takekawa, M., and Saito, H. (1998). Cell 95, 521–530. Tissir, F., and Goffinet, A.M. (2003). Nat. Rev. Neurosci. 4, 496–505. Vadlamudi, R.K., Li, F., Adam, L., Nguyen, D., Ohta, Y., Stossel, T.P., and Kumar, R. (2002). Nat. Cell Biol. 4, 681–690. Webb, D.J., Donais, K., Whitmore, L.A., Thomas, S.M., Turner, C.E., Parsons, J.T., and Horwitz, A.F. (2004). Nat. Cell Biol. 6, 154–161.
TIRFing out Studies on Glut4 Trafficking Xiao-Wei Chen1 and Alan R. Saltiel1,* 1
Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA *Correspondence: [email protected] DOI 10.1016/j.devcel.2006.12.008
Docking and fusion of Glut4 vesicles with the plasma membrane are essential but poorly understood steps during insulin-stimulated glucose transport. Recent studies utilizing TIRF microscopy shed light on these processes and map the sites of possible intervention by insulin from just underneath the plasma membrane.
Insulin stimulates glucose uptake into muscle and adipose cells by promoting the relocation of the glucose transporter Glut4 to the cell surface (Bryant et al., 2002). Despite the general notion that deficiencies in this process represent a primary lesion in the development of type 2 diabetes and related metabolic disorders, little is known about the molecular defects that underlie this pathology (Saltiel and Kahn, 2001). This is largely due to our limited knowledge of precisely how
insulin signaling influences the trafficking itinerary of Glut4. In the basal state, Glut4 is retained in intracellular vesicles and then traffics to the plasma membrane upon insulin stimulation. This process involves multiple steps governed by insulin, including endocytosis into endosomes, sorting of Glut4 into specialized storage vesicles, their retention inside cells, transport along cytoskeletal tracks, tethering, docking, priming, and the final fusion with the plasma
4 Developmental Cell 12, January 2007 ª2007 Elsevier Inc.
membrane. However, the regulated steps in this complex process that are rate limiting, let alone the precise identity of the molecular targets that connect insulin signaling to cellular vesicle trafficking machineries, have yet to be defined. Bai et al. (2007) have investigated the terminal stages of Glut4 trafficking by examining the motion of eGFP-tagged Glut4 molecules using time-lapsed total internal reflection fluorescence (TIRF) microscopy. This approach
Developmental Cell
Previews allows for the selective capture of the docking and fusion events that take place adjacent to the plasma membrane. They observed what appears to be a common pattern for Glut4 vesicle motion during the final stages of its journey to the plasma membrane. Some vesicles, referred to as docked vesicles, seem to remain static within a short range near the plasma membrane for a period (dwell time). Others appeared highly mobile and were thus defined as a part of the predocking stage. Finally, they detected the irreversible movements of some Glut4 vesicles toward the plasma membrane, and they considered these fusing vesicles. Intriguingly, insulin treatment of cells substantially accelerated the fusion rate (8-fold) of Glut4 vesicles with the plasma membrane, but led to only a moderate increase (2 fold) in their docking rate. These data were interpreted to indicate that fusion of Glut4 represented the major site at which insulin acts. In addition, the Akt substrate AS160 (Watson and Pessin, 2006) appears not to participate in this process. Despite some differences with two other recent studies (Gonzalez and McGraw, 2006; Lizunov et al., 2005), the current report is in agreement with the hypothesis that the terminal stages play a pivotal role in regulating Glut4 trafficking. Does this kind of analysis help in our understanding of the steps crucial to insulin action and how these events might be attenuated in insulin resistance? The focus on the final events in Glut4 docking and fusion is a characteristic of TIRF microscopy and allows for a careful examination in real time of
how the transporter finally achieves exposure to the extracellular space. Unfortunately, this approach does not permit assessment of the endocytosis and sorting of Glut4, nor its trafficking from perinuclear regions in the cell, all events that are regulated by insulin and thus potential defects in insulinresistant states. On the other hand, these analyses may now allow investigators to consider many more questions about the process. For example, how are the final stages connected to the penultimate steps in Glut4 vesicle trafficking? Although these earlier steps might not be as responsive to insulin, they may be even more important for building up a reservoir pool of Glut4 vesicles, as studies in other trafficking processes have indicated (Sudhof, 2004). How are Glut4 vesicles selectively presented to the fusion sites, since the SNARE and accessory proteins may not be sufficient to provide this specificity? Vesicle tethering and docking have been suggested to ensure fidelity of trafficking processes and perhaps directly facilitate the fusion events (Munson and Novick, 2006). In the case of insulin-stimulated glucose transport, these events might be carried out by the exocyst complex (Inoue et al., 2003). Hence, the exact links that escort Glut4 vesicles between docking and fusion have to be established. Perhaps the most important question centers on the molecular targets of insulin signaling that choreograph the entire program of Glut4 trafficking. Candidates emerging from recent studies include Rab proteins, SNARE proteins, the exocyst complex, and the
myosin motor Myo1c. Each of these appear to respond to a variety of different signaling events from the insulin receptor, such as phosphorylation, activation of guanyl nucleotide exchange factors and GTPase-activating proteins, and various polyphosphoinositides (Ramm and James, 2005; Watson et al., 2004). It will be of interest to test whether and how these signaling and structural proteins influence the dynamics of the terminal stages of Glut4 trafficking. The new advances in TIRF microscopy may facilitate our efforts to decipher how insulin orchestrates Glut4 translocation. REFERENCES Bai, L., Wang, Y., Fan, J., Chen, Y., Ji, W., Qu, A., Xu, P., James, D.E., and Xu, T. (2007). Cell Metab. 5, 47–51. Bryant, N.J., Govers, R., and James, D.E. (2002). Nat. Rev. Mol. Cell Biol. 3, 267–277. Gonzalez, E., and McGraw, T.E. (2006). Mol. Biol. Cell 17, 4484–4493. Inoue, M., Chang, L., Hwang, J., Chiang, S.H., and Saltiel, A.R. (2003). Nature 422, 629–633. Lizunov, V.A., Matsumoto, H., Zimmerberg, J., Cushman, S.W., and Frolov, V.A. (2005). J. Cell Biol. 169, 481–489. Munson, M., and Novick, P. (2006). Nat. Struct. Mol. Biol. 13, 577–581. Ramm, G., and James, D.E. (2005). Cell Metab. 2, 150–152. Saltiel, A.R., and Kahn, C.R. (2001). Nature 414, 799–806. Sudhof, T.C. (2004). Annu. Rev. Neurosci. 27, 509–547. Watson, R.T., and Pessin, J.E. (2006). Trends Biochem. Sci. 31, 215–222. Watson, R.T., Kanzaki, M., and Pessin, J.E. (2004). Endocr. Rev. 25, 177–204.
Developmental Cell 12, January 2007 ª2007 Elsevier Inc. 5