Encalada SE, Szpankowski L, Xia CH, Goldstein LS.
Stable kinesin and dynein assemblies drive the axonal transport of mammalian prion protein vesicles.
Cell. 2011 Feb 18;144(4):551-65.
PubMed.
With their recent paper on the transport of prion protein-containing vesicles, Encalada and her collaborators from Larry Goldstein’s laboratory made big steps in the right direction toward explaining how directionality of transport along axonal microtubules might be regulated in mammalian neurons. For us, "the right direction" means the right choice of the system of study and the tools employed. Few will disagree with the idea that transport is best studied by directly visualizing—in real time—the vesicles in motion. Also, few will disagree with the idea that this visualization should be done with minimal disruption of the intracellular environment. Practically, these two conditions are difficult to reconcile. First, visualization of a protein in motion implies that it is expressed as fusion protein, tagged with a fluorescent tracer, usually one of the many green fluorescent protein variants available today. In most cases, both the exogenous expression and the tag create problems. Ideally, one should look at the transport of an endogenous protein by the endogenous transport machinery, under native conditions. Transfection usually leads to expression of the tagged protein at above-normal levels, saturating the transport machinery, and the tag may interfere with the recruitment of the motors to the transport vesicle. Encalada et al. circumvent both problems by cleverly choosing the cellular prion protein (PrPC) as the prototype cargo they want to visualize. PrPC is a GPI-anchored glycoprotein, transported along the secretory pathway inside the carrier vesicles; this means that the tag—yellow fluorescent protein (YFP)—is also inside the lumen of the vesicle. In this way, the cytoplasmic face of the vesicle, which interacts with the molecular motor complexes, is not altered in any major way. The second clever thing that Encalada et al. do is to study the recruitment of the endogenous motors to the transport vesicles. Yes, they analyze motility of YFP-tagged PrPC with kymographs, but they use "vesicle mapping"—essentially immunocytochemistry of fixed specimens—to assess (literally, quantify) the number of motors associated with each vesicle, after prior recording of vesicle motility. The lesson from this study is that one should never underestimate the power of immunocytochemistry as a quantitative tool, and to work with endogenous, rather than expressed, proteins.
The focus of these experiments was not to study transport of PrPC, but to address the question of how directionality of cargo vesicle transport along microtubules is regulated. In axons, this refers to the regulation of anterograde—plus-end-directed—versus retrograde—minus-end-directed—motility. Nerve ligation experiments undoubtedly indicate that the vesicles containing the PrPC move overall anterogradely, toward axon terminals. Yet, en route to the synapse, over shorter periods of time, many of them also move retrogradely, switching between anterograde and retrograde movement. Interestingly, Encalada et al. find that this switch in the direction of movement is enabled not by alternative release and recruitment of motors of opposed directionality, but by modulating, in a coordinated fashion, the activity of kinesin-1, the plus-end motor, and of cytoplasmic dynein, the minus-end motor, while both remain attached to the vesicle. Thus, the activities of kinesin-1 and cytoplasmic dynein seem tightly coupled, and the inactivation of one motor or another—done in this study with RNAi or with gene-targeted deletions of motor subunits—changes the direction of movement in unpredicted fashions. For example, the disruption of kinesin-1 not only decreases anterograde motility, but also the retrograde motility of PrPC-containing vesicles. This unexpected result contradicts the proposed tug-of-war mechanism, where the direction of vesicle movement is determined by the "stronger" team of opposing vesicle motors. It is not yet clear how the simultaneous regulation of the activities of the two types of motors is achieved. Encalada et al. envision some sort of physical interaction between kinesin-1 and cytoplasmic dynein. In our view, it is likely that scaffolding proteins capable of simultaneously binding kinesin-1 and cytoplasmic dynein could play a role. One such adaptor could be the Jun kinase (JNK)-interacting protein-1 (JIP-1), which enables the recruitment of kinesin-1 to vesicles carrying another famous cargo protein, the amyloid-β precursor protein (APP) (1,2). Recently, Erika Holzbaur’s group at the University of Pennsylvania showed that JIP-1 also binds a subunit of dynactin (3), an accessory protein complex of cytoplasmic dynein. Also, the C. elegans homologue of another JNK-interacting protein, JIP-3, was shown to bind both kinesin-1 and cytoplasmic dynein, enabling their co-transport into axons (4). Although vesicle association of this tripartite complex was not reported, we previously showed that JIP-3 is associated with cargo vesicles in mammalian neurons (5).
On the same note, DISC1, a protein shown to bind at the same time kinesin-1 and cytoplasmic dynein (6), could be an example of an adaptor protein that recruits antagonistic molecular motors to the same vesicle. Intriguingly, DISC1 seems to be implicated in the transport of the APP-derived, amyloid-β (Aβ) peptide (7,8), a cargo that—like the prion protein studied by Goldstein’s group—is invisible to cytoplasmic proteins, being encapsulated within the carrier vesicle. Aβ is possibly tethered to the inner leaflet of the vesicle membrane via hydrophobic interactions. It may turn out that, rather than being passive scaffolds that recruit the motors to transport vesicles, adapter proteins such as JIP-1, JIP-3, or DISC1 could also regulate the coordinated activation and inactivation of the motors. The detailed molecular mechanisms that regulate the coordination and cooperation between motors of opposite direction of movement during vesicle transport remain to be elucidated. Certainly, such regulation is essential to allow the delivery of specific cargoes to their destination precisely when they are needed.
Whether the mechanism of regulation of directionality of transport described by Encalada et al. also applies to vesicles transporting other cargoes remains to be seen. Override (9) and tug-of-war, mechanisms (10) could regulate transport of other cargo vesicles, or may operate in other cell types. As for the relevance for prion disease, and for neurodegenerative diseases in general, the contribution of the study of Encalada et al. is enormous. While it remains to be determined whether a deficient axonal transport is a cause, a contributing factor, or a consequence of the neuronal pathology in these diseases, there is no question that axonal transport is indeed abnormal in many neurodegenerative diseases (11). What is deregulated (e.g., motor recruitment to cargo, motor activation, their coordinated function, or the microtubule tracks), and how could the abnormality be corrected will only be known from studies like the one by Encalada and collaborators.
References: See also Fu, M. and E.L. Holzbaur, JNK-induced changes in axonal transport are mediated by the bidirectional co-regulator JIP1. 50th Annual Meeting of the American Society for Cell Biology, Philadelphia, December 11-15, 2010; Muresan, V., B.T. Lamb, and Z. Muresan, DISC1 is required for the formation of intracellular Abeta oligomers, suggesting a link between schizophrenia and Alzheimer’s disease. Annual Meeting of the Society for Neuroscience, San Diego, November 13-17, 2010; and Muresan, V. and Z. Muresan, Disrupted-in-Schizophrenia 1 (DISC1) Facilitates the Intracellular Generation and Aggregation of Amyloid-beta (Abeta): A Link between Schizophrenia and Alzheimer’s Disease. 50th Annual Meeting of the American Society for Cell Biology, Philadelphia, December 11-15, 2010.
References:
Matsuda S, Matsuda Y, D'Adamio L.
Amyloid beta protein precursor (AbetaPP), but not AbetaPP-like protein 2, is bridged to the kinesin light chain by the scaffold protein JNK-interacting protein 1.
J Biol Chem. 2003 Oct 3;278(40):38601-6.
PubMed.
Muresan Z, Muresan V.
Coordinated transport of phosphorylated amyloid-beta precursor protein and c-Jun NH2-terminal kinase-interacting protein-1.
J Cell Biol. 2005 Nov 21;171(4):615-25.
PubMed.
Arimoto M, Koushika SP, Choudhary BC, Li C, Matsumoto K, Hisamoto N.
The Caenorhabditis elegans JIP3 protein UNC-16 functions as an adaptor to link kinesin-1 with cytoplasmic dynein.
J Neurosci. 2011 Feb 9;31(6):2216-24.
PubMed.
Muresan Z, Muresan V.
c-Jun NH2-terminal kinase-interacting protein-3 facilitates phosphorylation and controls localization of amyloid-beta precursor protein.
J Neurosci. 2005 Apr 13;25(15):3741-51.
PubMed.
Taya S, Shinoda T, Tsuboi D, Asaki J, Nagai K, Hikita T, Kuroda S, Kuroda K, Shimizu M, Hirotsune S, Iwamatsu A, Kaibuchi K.
DISC1 regulates the transport of the NUDEL/LIS1/14-3-3epsilon complex through kinesin-1.
J Neurosci. 2007 Jan 3;27(1):15-26.
PubMed.
Muresan V, Godek CP, Reese TS, Schnapp BJ.
Plus-end motors override minus-end motors during transport of squid axon vesicles on microtubules.
J Cell Biol. 1996 Oct;135(2):383-97.
PubMed.
Hendricks AG, Perlson E, Ross JL, Schroeder HW, Tokito M, Holzbaur EL.
Motor coordination via a tug-of-war mechanism drives bidirectional vesicle transport.
Curr Biol. 2010 Apr 27;20(8):697-702.
PubMed.
Muresan V, Muresan Z.
Is abnormal axonal transport a cause, a contributing factor or a consequence of the neuronal pathology in Alzheimer's disease?.
Future Neurol. 2009 Nov 1;4(6):761-773.
PubMed.
Comments
Rutgers - New Jersey Medical School
With their recent paper on the transport of prion protein-containing vesicles, Encalada and her collaborators from Larry Goldstein’s laboratory made big steps in the right direction toward explaining how directionality of transport along axonal microtubules might be regulated in mammalian neurons. For us, "the right direction" means the right choice of the system of study and the tools employed. Few will disagree with the idea that transport is best studied by directly visualizing—in real time—the vesicles in motion. Also, few will disagree with the idea that this visualization should be done with minimal disruption of the intracellular environment. Practically, these two conditions are difficult to reconcile. First, visualization of a protein in motion implies that it is expressed as fusion protein, tagged with a fluorescent tracer, usually one of the many green fluorescent protein variants available today. In most cases, both the exogenous expression and the tag create problems. Ideally, one should look at the transport of an endogenous protein by the endogenous transport machinery, under native conditions. Transfection usually leads to expression of the tagged protein at above-normal levels, saturating the transport machinery, and the tag may interfere with the recruitment of the motors to the transport vesicle. Encalada et al. circumvent both problems by cleverly choosing the cellular prion protein (PrPC) as the prototype cargo they want to visualize. PrPC is a GPI-anchored glycoprotein, transported along the secretory pathway inside the carrier vesicles; this means that the tag—yellow fluorescent protein (YFP)—is also inside the lumen of the vesicle. In this way, the cytoplasmic face of the vesicle, which interacts with the molecular motor complexes, is not altered in any major way. The second clever thing that Encalada et al. do is to study the recruitment of the endogenous motors to the transport vesicles. Yes, they analyze motility of YFP-tagged PrPC with kymographs, but they use "vesicle mapping"—essentially immunocytochemistry of fixed specimens—to assess (literally, quantify) the number of motors associated with each vesicle, after prior recording of vesicle motility. The lesson from this study is that one should never underestimate the power of immunocytochemistry as a quantitative tool, and to work with endogenous, rather than expressed, proteins.
The focus of these experiments was not to study transport of PrPC, but to address the question of how directionality of cargo vesicle transport along microtubules is regulated. In axons, this refers to the regulation of anterograde—plus-end-directed—versus retrograde—minus-end-directed—motility. Nerve ligation experiments undoubtedly indicate that the vesicles containing the PrPC move overall anterogradely, toward axon terminals. Yet, en route to the synapse, over shorter periods of time, many of them also move retrogradely, switching between anterograde and retrograde movement. Interestingly, Encalada et al. find that this switch in the direction of movement is enabled not by alternative release and recruitment of motors of opposed directionality, but by modulating, in a coordinated fashion, the activity of kinesin-1, the plus-end motor, and of cytoplasmic dynein, the minus-end motor, while both remain attached to the vesicle. Thus, the activities of kinesin-1 and cytoplasmic dynein seem tightly coupled, and the inactivation of one motor or another—done in this study with RNAi or with gene-targeted deletions of motor subunits—changes the direction of movement in unpredicted fashions. For example, the disruption of kinesin-1 not only decreases anterograde motility, but also the retrograde motility of PrPC-containing vesicles. This unexpected result contradicts the proposed tug-of-war mechanism, where the direction of vesicle movement is determined by the "stronger" team of opposing vesicle motors. It is not yet clear how the simultaneous regulation of the activities of the two types of motors is achieved. Encalada et al. envision some sort of physical interaction between kinesin-1 and cytoplasmic dynein. In our view, it is likely that scaffolding proteins capable of simultaneously binding kinesin-1 and cytoplasmic dynein could play a role. One such adaptor could be the Jun kinase (JNK)-interacting protein-1 (JIP-1), which enables the recruitment of kinesin-1 to vesicles carrying another famous cargo protein, the amyloid-β precursor protein (APP) (1,2). Recently, Erika Holzbaur’s group at the University of Pennsylvania showed that JIP-1 also binds a subunit of dynactin (3), an accessory protein complex of cytoplasmic dynein. Also, the C. elegans homologue of another JNK-interacting protein, JIP-3, was shown to bind both kinesin-1 and cytoplasmic dynein, enabling their co-transport into axons (4). Although vesicle association of this tripartite complex was not reported, we previously showed that JIP-3 is associated with cargo vesicles in mammalian neurons (5).
On the same note, DISC1, a protein shown to bind at the same time kinesin-1 and cytoplasmic dynein (6), could be an example of an adaptor protein that recruits antagonistic molecular motors to the same vesicle. Intriguingly, DISC1 seems to be implicated in the transport of the APP-derived, amyloid-β (Aβ) peptide (7,8), a cargo that—like the prion protein studied by Goldstein’s group—is invisible to cytoplasmic proteins, being encapsulated within the carrier vesicle. Aβ is possibly tethered to the inner leaflet of the vesicle membrane via hydrophobic interactions. It may turn out that, rather than being passive scaffolds that recruit the motors to transport vesicles, adapter proteins such as JIP-1, JIP-3, or DISC1 could also regulate the coordinated activation and inactivation of the motors. The detailed molecular mechanisms that regulate the coordination and cooperation between motors of opposite direction of movement during vesicle transport remain to be elucidated. Certainly, such regulation is essential to allow the delivery of specific cargoes to their destination precisely when they are needed.
Whether the mechanism of regulation of directionality of transport described by Encalada et al. also applies to vesicles transporting other cargoes remains to be seen. Override (9) and tug-of-war, mechanisms (10) could regulate transport of other cargo vesicles, or may operate in other cell types. As for the relevance for prion disease, and for neurodegenerative diseases in general, the contribution of the study of Encalada et al. is enormous. While it remains to be determined whether a deficient axonal transport is a cause, a contributing factor, or a consequence of the neuronal pathology in these diseases, there is no question that axonal transport is indeed abnormal in many neurodegenerative diseases (11). What is deregulated (e.g., motor recruitment to cargo, motor activation, their coordinated function, or the microtubule tracks), and how could the abnormality be corrected will only be known from studies like the one by Encalada and collaborators.
References: See also Fu, M. and E.L. Holzbaur, JNK-induced changes in axonal transport are mediated by the bidirectional co-regulator JIP1. 50th Annual Meeting of the American Society for Cell Biology, Philadelphia, December 11-15, 2010; Muresan, V., B.T. Lamb, and Z. Muresan, DISC1 is required for the formation of intracellular Abeta oligomers, suggesting a link between schizophrenia and Alzheimer’s disease. Annual Meeting of the Society for Neuroscience, San Diego, November 13-17, 2010; and Muresan, V. and Z. Muresan, Disrupted-in-Schizophrenia 1 (DISC1) Facilitates the Intracellular Generation and Aggregation of Amyloid-beta (Abeta): A Link between Schizophrenia and Alzheimer’s Disease. 50th Annual Meeting of the American Society for Cell Biology, Philadelphia, December 11-15, 2010.
References:
Matsuda S, Matsuda Y, D'Adamio L. Amyloid beta protein precursor (AbetaPP), but not AbetaPP-like protein 2, is bridged to the kinesin light chain by the scaffold protein JNK-interacting protein 1. J Biol Chem. 2003 Oct 3;278(40):38601-6. PubMed.
Muresan Z, Muresan V. Coordinated transport of phosphorylated amyloid-beta precursor protein and c-Jun NH2-terminal kinase-interacting protein-1. J Cell Biol. 2005 Nov 21;171(4):615-25. PubMed.
Arimoto M, Koushika SP, Choudhary BC, Li C, Matsumoto K, Hisamoto N. The Caenorhabditis elegans JIP3 protein UNC-16 functions as an adaptor to link kinesin-1 with cytoplasmic dynein. J Neurosci. 2011 Feb 9;31(6):2216-24. PubMed.
Muresan Z, Muresan V. c-Jun NH2-terminal kinase-interacting protein-3 facilitates phosphorylation and controls localization of amyloid-beta precursor protein. J Neurosci. 2005 Apr 13;25(15):3741-51. PubMed.
Taya S, Shinoda T, Tsuboi D, Asaki J, Nagai K, Hikita T, Kuroda S, Kuroda K, Shimizu M, Hirotsune S, Iwamatsu A, Kaibuchi K. DISC1 regulates the transport of the NUDEL/LIS1/14-3-3epsilon complex through kinesin-1. J Neurosci. 2007 Jan 3;27(1):15-26. PubMed.
Muresan V, Godek CP, Reese TS, Schnapp BJ. Plus-end motors override minus-end motors during transport of squid axon vesicles on microtubules. J Cell Biol. 1996 Oct;135(2):383-97. PubMed.
Hendricks AG, Perlson E, Ross JL, Schroeder HW, Tokito M, Holzbaur EL. Motor coordination via a tug-of-war mechanism drives bidirectional vesicle transport. Curr Biol. 2010 Apr 27;20(8):697-702. PubMed.
Muresan V, Muresan Z. Is abnormal axonal transport a cause, a contributing factor or a consequence of the neuronal pathology in Alzheimer's disease?. Future Neurol. 2009 Nov 1;4(6):761-773. PubMed.
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