Browsing by Subject "Dynamins"
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Item Dynamin 2 Mutations Implicated in Charcot-Marie-Tooth Disease(2010-05-14) Tassin, Tara Charisse; Albanesi, Joseph P.Dynamins are large (100 kDa) GTPases responsible for severing the necks of nascent vesicles during clathrin- and caveolae-mediated endocytosis, and are implicated in a variety of other cellular processes, including macropinocytosis, phagocytosis, and cytoskeletal organization. Mammalian cells contain three dynamin genes, encoding dynamin 1 (expressed in neurons and neuroendocrine cells), dynamin 2 (ubiquitously expressed), and dynamin 3 (enriched in testes, but also found in pre- and post-synaptic regions of neurons). Dynamin 2 was identified as a locus for Charcot-Marie-Tooth disease (CMT) and Centronuclear Myopathy (CNM). CMT is a peripheral neuropathy affecting 1 in every 2,500 people, making it one of the most commonly inherited neurological disorders. CNM causes progressive loss of muscle tone without primary neuronal involvement. In this study, the effects of two CMT mutations were characterized in order to gain insight into the causes of the disease. The two mutations, K558E and delDEE (a deletion of residues 551-553), are both located in the Pleckstrin Homology (PH) domain (approximately residues 520-630), which mediates the binding of dynamins to phosphoinositide lipids and to βγ subunits of heterotrimeric G proteins. The overall goal of the project was to determine how the mutations influence fundamental properties of dynamin 2, including: 1. Self-assembly and concentration-dependent GTPase activation; 2. Binding to phosphatidylinositol-(4,5)- bisphosphate (hereafter termed PIP2) and stimulation of GTPase activity by PIP2; 3. Stimulus-dependent tyrosine phosphorylation; and 4. Interaction with G-βγ. In summary, I have found that Dyn 2-K558E undergoes normal self-assembly and self-activation, but that its activation by PIP2-containing vesicles is drastically reduced. Consistent with this observation, the ability of the isolated K558E PH domain to bind to PIP2-containing vesicles was also impaired. Because full-length Dyn 2-delDEE could not be expressed in Sf9 cells, I was unable to determine effects of this deletion on its self-assembly, self-activation, or activation by PIP2 vesicles. However, I took advantage of the bacterially-expressed GST-tagged PH domain to demonstrate that deletion of residues DEE does not affect binding to PIP2, whereas it strongly (6-20 fold) enhances the interaction with G-βγ. This enhanced binding may be significant in explaining the role of the delDEE mutation in CMT disease, as previous studies have shown that G-βγ inhibits the GTPase activity of dynamin 1. Although full-length Dyn 2-delDEE protein could not be obtained for in vitro analysis, I was able to express the full-length mutant in mammalian cells, allowing me to examine its ability to undergo tyrosine phosphorylation. Consistently, Dyn 2-delDEE underwent approximately 2-3 fold higher levels of tyrosine phosphorylation than either wild-type dynamin 2 or Dyn 2-K558E in Src-expressing cells stimulated by EGF or isoproterenol. Mutation of the two tyrosines (individually or in combination) previously shown to be the major Src-phosphorylated sites in dynamins significantly reduced tyrosine phosphorylation in both wild-type and mutant dynamins. Finally, I compared the effects of overexpression of wild-type dynamin 2 and Dyn 2-delDEE on stimulus-dependent activation of the MAP kinases Erk1/2. These experiments were motivated by earlier studies indicating that maximal Erk activation cannot occur if receptor-mediated endocytosis is inhibited. Overexpression of Dyn 2-delDEE reduced Erk activation by 70%, and activation was further reduced by mutation of the two phosphorylatable tyrosines. Mutation of the phosphorylatable tyrosines in wild-type dynamin 2 resulted in a 50% inhibition of Erk activation. Overall, the results of my analysis demonstrate that two CMT mutations within the same domain of dynamin 2 have distinctly different properties. Future studies will be aimed at determining if these mutants impair endocytosis by distinct mechanisms.Item Genetic Dissection of Synaptic Vesicle Endocytosis(2019-03-28) Afuwape, Olusoji Adeyemi; Terman, Jonathan R.; Kavalali, Ege T.; Schmid, Sandra; Monteggia, Lisa; Krämer, HelmutSynaptic transmission is mediated by the quantal release of neurotransmitters through the fusion of discrete synaptic vesicles with the presynaptic membrane. To maintain reliable transmission, synaptic vesicles and proteins must be recycled after release of neurotransmitters. A key protein in this recycling process is dynamin. Dynamin is a GTPase that catalyzes the scission of a budding endosome off its parent membrane. The mammalian brain expresses three isoforms of dynamin. Using genetically modified mouse hippocampal neurons, I analyzed the functional significance of dynamin in synaptic vesicle endocytosis. Specifically, I assessed dynamin 2 function in synaptic vesicle recycling and neurotransmission and investigated the role of dynamin independent endocytosis at the synapse. My data demonstrates that synaptic transmission after post-natal knockout of dynamin 2 remains intact and synaptic vesicle endocytosis, assessed by the trafficking of vesicular glutamate transporter fused to pHluorin, is unperturbed. Synaptic vesicle endocytosis in the absence of dynamin 2 was assessed at both room temperature and 32 oC. At both temperatures, my results reveal that synaptic vesicle recycling functions independent of dynamin 2 but also, the kinetics of single vesicle recycling is unaffected by changes in temperature suggesting that a single, temperature insensitive (within the limits of testing) form of endocytosis mediates single synaptic vesicle endocytosis. Further experiments reveal that the retrieval of single synaptic vesicles persists after the knockout of all dynamin isoforms. However, after multivesicular release, my results show an overall decrease in synaptic vesicle pool size and a retardation of subsequent vesicle endocytosis in neurons lacking all dynamin isoforms suggesting dynamin function at the synapse is activity dependent. This finding is consistent with prior reports showing dynamin function at the synapse is dependent on its dephosphorylation by the Ca2= dependent phosphatase, calcineurin. My results also demonstrate a dichotomy in the dependence of dynamin for synaptic neurotransmission. Whereas I observe a decrease in evoked amplitude, release probability and frequency of spontaneously released events in glutamatergic synapses, I observe no discernable defects in GABAergic neurotransmission. This result suggests inhibitory synapses are better equipped with compensatory mechanisms to deal with the loss of dynamins 1,2 and 3. Overall, my data demonstrate that dynamin is crucial but not essential for synaptic vesicle endocytosis. Dynamin is an activity dependent GTPase that is required for synaptic vesicle recycling after exocytosis of multiple vesicles. However, the underlying mechanism of single synaptic vesicle endocytosis is both dynamin and temperature independent.