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PKA: Dynamic Assembly and Regulation of Macromolecular Signaling Complexes
Saturday, February 11, 2017 include myosin-Is, which are the widely expressed members of the myosin superfamily that bind directly to membranes, linking them to the actin cytoskeleton. To better understand the molecular roles of myosin-I isoforms, and the mechanisms by which they modulate membrane dynamics, we performed a range of single-molecule biophysical, structural, biochemical, and cell biological experiments to determine force-generating, membrane-binding, and motile properties of these motors. We discovered that there is remarkable diversity among myosin-I isoforms in their ability to alter their ATPase activities in response to mechanical forces, and we are learning how structural features within the motor domain lead to chemomechanical tuning. We have also learned much about the biophysics of the attachment of myosinI to membrane bilayers in the presence and absence of mechanical load. Our understanding of the biophysical properties of myosin-I isoforms has allowed us to further explore their roles in membrane dynamics via multimotor in vitro assays that reconstitute membrane motility and tubulation. This work is supported by grants from NIH/NIGMS (GM057247 and GM087253).
Exocytosis & Endocytosis Subgroup 59-Subg How Voltage-Gated Cav1 L-Type Ca2D Channels Meet the Needs of the Ribbonsynapse Amy Lee. Molecular Physiology & Biophysics, University of Iowa, Iowa City, IA, USA. Unlike the predominance of presynaptic Cav2 (P/Q- and N-type) Ca2þ channels most synapses, Cav1.3 and Cav1.4 L-type Ca2þ channels mediate exocytosis at ribbon synapses formed by inner hair cells in the cochlea and photoreceptors in the retina. Cav1.3 and Cav1.4 exhibit distinct biophysical properties from their Cav2 counterparts. At the ribbon synapse, Cav1.3 and Cav1.4 channels undergo little Ca2þ-dependent inactivation, which helps support the sustained release of glutamate required for proper encoding of sensory information. This is accomplished by different forms of channel regulation in inner hair cells and photoreceptors, which are disrupted by mutations causing disorders of hearing and vision in humans. In addition to their exocytotic function, Cav1.4 channels also regulate the formation and maintenance of the photoreceptor synapse in ways that are in part, independent of their Ca2þ conducting function. Our results highlight the diverse mechanisms by which Cav1 channels have taken on their unique roles at sensory ribbon synapses. 60-Subg Presynaptic Membrane Turnover and Transmitter Release at the Calyx of Held Xuelin Lou. Dept. Neuroscience, University of Wisconsin, Madison, WI, USA. Presynaptic neurotransmitter release parallels vigorous vesicle recycling, by which synapses ensure efficient vesicle replenishment, release site clearance, protein sorting, and structural integrity. Four membrane retrieval pathways have been proposed at nerve terminals, but controversy remains regarding the endocytic modes, underlying molecules, and physiological functions despite decades of tremendous effort. In particular, the recent findings on ultrafast endocytosis and dynamin-independent vesicle generation from rapidly retrieved bulk membrane challenge the previous recycling diagram and raise many interesting new questions. Moreover, increasing evidence suggests several important roles of endocytosis during fast synaptic transmission, in addition to its well-characterized function in vesicle resupply. Dynamin is a large GTPase, and it is required for clathrin-mediate endocytosis and maintaining readily releasable vesicle pool at synapses. Perturbations of this fission machinery offer a useful way to gain insights into cell physiology of membrane turnover at central synapses. Here, we summarize our recent work at the calyx of Held synapse, a fast glutamatergic terminal in the auditory brainstem. We found that multiple dynamin isoforms co-express at the calyx of Held and show isoform specific up regulation during development. Depending on the synaptic activity, dynamin-mediated endocytosis is required for several synaptic functions including vesicle genesis, transmitter release, short-term plasticity, quantal size stability, and calyx structure formation in vivo. We will discuss the role of dynamin in synaptic development, vesicle exo-endocytosis coupling, and the potential feedback from endocytosis at endocytic zones to exocytosis at active zones during fast transmitter release.
11a
61-Subg The Long Road to Micro-Dynamic Presynaptic FRET Measurements Robert Zucker. Dept. Molec/Cell Biol, University of California, Berkeley, Berkeley, CA, USA. I will review the history and lead-up to our attempt to measure changes in FRET in assembled SNARE complexes in a very small space and a very short time, where only a tiny fraction of each of the labeled SNARE proteins is part of a SNARE complex. After recounting the saga of grant support and technical development, I will summarize our main findings: Using donor dequenching by receptor bleach as well as FLIM, we find a resting FRET indicating a number of assembled SNARE complexes exceeding the expected fraction of synaptobrevins (or VAMPs) that are in assembled SNAREs of docked and primed vesicles, likely reflecting the existence of stillassembled ‘‘orphan SNAREs’’ left over from prior bouts of secretion. We can detect the dispersion of all three SNARE proteins – VAMP, SNAP-25, and syntaxin – as well as the FRETting complex of assembled SNAREs itself, from the center of the presynaptic active zone to the periphery during and after a train of action potentials. Using sensitized acceptor emission on donor excitation, we also detect the disassembly of N-terminally labeled SNAREs prior to endocytosis of vesicle membrane and proteins, and the assembly of new SNAREs as replacement vesicles dock and prime, and we occasionally see a transient FRET increase while vesicles fuse during the train. By donor quenching, we also detect the rearrangement of C-terminally labeled SNAREs when vesicles fuse with the plasma membrane, and their subsequent dispersion, disassembly, and re-assembly. A variety of control experiments and statistical tests rule out most sources of artefact and alternative interpretations. I will also speculate on why SNARES disperse and are disassembled in the periphery, rather than in the central active zone where exocytosis occurs, as we had originally expected.
Intrinsically Disordered Proteins Subgroup 62-Subg PKA: Dynamic Assembly and Regulation of Macromolecular Signaling Complexes Susan S. Taylor. Dept. of Pharmacology/ Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA. PKA Signaling: Linkers, Loops, and Intrinsically Disorder Regions Drive the Dynamic Assembly of Holoenzyme Complexes. The PKA catalytic (C) subunit crystallized in 1991, gave us our first glimpse of the fold that describes all protein kinases. The two lobes that comprise the core are flanked by N-and C-terminal tails, which wrap around both lobes. By capturing all stages of catalysis in crystal structures we appreciate the dynamic nature of these tails and how they undergo order-disorder transitions as part of the catalytic cycle. Ordering the activation loop by a critical phosphate is another fundamental order/disorder transition that is a conserved feature of most protein kinases. These dynamic features allow the kinases to function as highly regulated molecular switches. In the case of PKA, the C-subunit is packaged with regulatory (R) subunit dimers where each chain contains two C-terminal cAMP binding (CNB) domains. The CNB domains are highly dynamic allosteric signaling modules that have been conserved to translate a biological response to cAMP. Although the fundamental features of this domain were elucidated by structures of R-monomers and R:C heterodimers, it required full-length R2C2 tetramers to appreciate the importance of symmetry for PKA signaling. Each R-subunit contains an N-terminal dimerization domain that is also a docking site for scaffold proteins referred to as A Kinase Anchoring Proteins (AKAPs). This domain is joined by a flexible linker to the CNB domains, and embedded within the linker is an inhibitor site that docks to the active site of the C-subunit in the holoenzyme. The order/disorder transitions of the linker drive the assembly of the holoenzymes which each have distinct quaternary structures. The linkers, rich in biological information, are an essential feature of PKA regulation. (Funded by NIH GM34921 and DK54441.) 63-Subg Disordered Cdk Substrates Act as Multi-Input Signal Processors to Control the Key Decision Points in the Cell Cycle Mart Loog. University of Tartu, Tartu, Estonia. The decision points between different cell fates involve systems that process alternative signals into binary choices. At the beginning of the cell cycle the
Exocytosis & Endocytosis Subgroup 59-Subg How Voltage-Gated Cav1 L-Type Ca2D Channels Meet the Needs of the Ribbonsynapse Amy Lee. Molecular Physiology & Biophysics, University of Iowa, Iowa City, IA, USA. Unlike the predominance of presynaptic Cav2 (P/Q- and N-type) Ca2þ channels most synapses, Cav1.3 and Cav1.4 L-type Ca2þ channels mediate exocytosis at ribbon synapses formed by inner hair cells in the cochlea and photoreceptors in the retina. Cav1.3 and Cav1.4 exhibit distinct biophysical properties from their Cav2 counterparts. At the ribbon synapse, Cav1.3 and Cav1.4 channels undergo little Ca2þ-dependent inactivation, which helps support the sustained release of glutamate required for proper encoding of sensory information. This is accomplished by different forms of channel regulation in inner hair cells and photoreceptors, which are disrupted by mutations causing disorders of hearing and vision in humans. In addition to their exocytotic function, Cav1.4 channels also regulate the formation and maintenance of the photoreceptor synapse in ways that are in part, independent of their Ca2þ conducting function. Our results highlight the diverse mechanisms by which Cav1 channels have taken on their unique roles at sensory ribbon synapses. 60-Subg Presynaptic Membrane Turnover and Transmitter Release at the Calyx of Held Xuelin Lou. Dept. Neuroscience, University of Wisconsin, Madison, WI, USA. Presynaptic neurotransmitter release parallels vigorous vesicle recycling, by which synapses ensure efficient vesicle replenishment, release site clearance, protein sorting, and structural integrity. Four membrane retrieval pathways have been proposed at nerve terminals, but controversy remains regarding the endocytic modes, underlying molecules, and physiological functions despite decades of tremendous effort. In particular, the recent findings on ultrafast endocytosis and dynamin-independent vesicle generation from rapidly retrieved bulk membrane challenge the previous recycling diagram and raise many interesting new questions. Moreover, increasing evidence suggests several important roles of endocytosis during fast synaptic transmission, in addition to its well-characterized function in vesicle resupply. Dynamin is a large GTPase, and it is required for clathrin-mediate endocytosis and maintaining readily releasable vesicle pool at synapses. Perturbations of this fission machinery offer a useful way to gain insights into cell physiology of membrane turnover at central synapses. Here, we summarize our recent work at the calyx of Held synapse, a fast glutamatergic terminal in the auditory brainstem. We found that multiple dynamin isoforms co-express at the calyx of Held and show isoform specific up regulation during development. Depending on the synaptic activity, dynamin-mediated endocytosis is required for several synaptic functions including vesicle genesis, transmitter release, short-term plasticity, quantal size stability, and calyx structure formation in vivo. We will discuss the role of dynamin in synaptic development, vesicle exo-endocytosis coupling, and the potential feedback from endocytosis at endocytic zones to exocytosis at active zones during fast transmitter release.
11a
61-Subg The Long Road to Micro-Dynamic Presynaptic FRET Measurements Robert Zucker. Dept. Molec/Cell Biol, University of California, Berkeley, Berkeley, CA, USA. I will review the history and lead-up to our attempt to measure changes in FRET in assembled SNARE complexes in a very small space and a very short time, where only a tiny fraction of each of the labeled SNARE proteins is part of a SNARE complex. After recounting the saga of grant support and technical development, I will summarize our main findings: Using donor dequenching by receptor bleach as well as FLIM, we find a resting FRET indicating a number of assembled SNARE complexes exceeding the expected fraction of synaptobrevins (or VAMPs) that are in assembled SNAREs of docked and primed vesicles, likely reflecting the existence of stillassembled ‘‘orphan SNAREs’’ left over from prior bouts of secretion. We can detect the dispersion of all three SNARE proteins – VAMP, SNAP-25, and syntaxin – as well as the FRETting complex of assembled SNAREs itself, from the center of the presynaptic active zone to the periphery during and after a train of action potentials. Using sensitized acceptor emission on donor excitation, we also detect the disassembly of N-terminally labeled SNAREs prior to endocytosis of vesicle membrane and proteins, and the assembly of new SNAREs as replacement vesicles dock and prime, and we occasionally see a transient FRET increase while vesicles fuse during the train. By donor quenching, we also detect the rearrangement of C-terminally labeled SNAREs when vesicles fuse with the plasma membrane, and their subsequent dispersion, disassembly, and re-assembly. A variety of control experiments and statistical tests rule out most sources of artefact and alternative interpretations. I will also speculate on why SNARES disperse and are disassembled in the periphery, rather than in the central active zone where exocytosis occurs, as we had originally expected.
Intrinsically Disordered Proteins Subgroup 62-Subg PKA: Dynamic Assembly and Regulation of Macromolecular Signaling Complexes Susan S. Taylor. Dept. of Pharmacology/ Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA. PKA Signaling: Linkers, Loops, and Intrinsically Disorder Regions Drive the Dynamic Assembly of Holoenzyme Complexes. The PKA catalytic (C) subunit crystallized in 1991, gave us our first glimpse of the fold that describes all protein kinases. The two lobes that comprise the core are flanked by N-and C-terminal tails, which wrap around both lobes. By capturing all stages of catalysis in crystal structures we appreciate the dynamic nature of these tails and how they undergo order-disorder transitions as part of the catalytic cycle. Ordering the activation loop by a critical phosphate is another fundamental order/disorder transition that is a conserved feature of most protein kinases. These dynamic features allow the kinases to function as highly regulated molecular switches. In the case of PKA, the C-subunit is packaged with regulatory (R) subunit dimers where each chain contains two C-terminal cAMP binding (CNB) domains. The CNB domains are highly dynamic allosteric signaling modules that have been conserved to translate a biological response to cAMP. Although the fundamental features of this domain were elucidated by structures of R-monomers and R:C heterodimers, it required full-length R2C2 tetramers to appreciate the importance of symmetry for PKA signaling. Each R-subunit contains an N-terminal dimerization domain that is also a docking site for scaffold proteins referred to as A Kinase Anchoring Proteins (AKAPs). This domain is joined by a flexible linker to the CNB domains, and embedded within the linker is an inhibitor site that docks to the active site of the C-subunit in the holoenzyme. The order/disorder transitions of the linker drive the assembly of the holoenzymes which each have distinct quaternary structures. The linkers, rich in biological information, are an essential feature of PKA regulation. (Funded by NIH GM34921 and DK54441.) 63-Subg Disordered Cdk Substrates Act as Multi-Input Signal Processors to Control the Key Decision Points in the Cell Cycle Mart Loog. University of Tartu, Tartu, Estonia. The decision points between different cell fates involve systems that process alternative signals into binary choices. At the beginning of the cell cycle the