Biology subjects

Vevea, J. D.

Publications and source records attributed to Vevea, J. D..

5 recordsLinked to original sources

bioRxiv2022

Mitochondrial complementation occurs in rat hippocampal axons and supports the synaptic vesicle cycle.

Mitochondria exert powerful control over cellular physiology, contributing to ion homeostasis, energy production, and metabolite biosynthesis. Mitochondrial trafficking and function are vital to neurons, with organelle impairment or altered morphology observed in every neurodegenerative disorder studied. While this organelle and its biosynthetic products are critical for cellular function, decreased output and/or byproducts (e.g., free radicals), can be harmful, and organelle quality control (QC) mechanisms are required to maintain function and prevent a cascade of damage. Owing to its length and general lack of biosynthetic machinery, the axon is particularly sensitive to damage and there is little consensus regarding the details of mitochondrial QC mechanisms in this compartment. Here we investigate the basal, unstressed behavior of axonal mitochondria, focusing on mitochondrial trafficking and fusion to better understand potential QC mechanisms. We observed size and redox asymmetry of mitochondrial traffic in the axon, suggestive of an active QC mechanism. Importantly, we demonstrate, in detail, biochemical complementation of axonal mitochondria. Upon disruption of mitochondrial fusion, we observed an altered synaptic proteome, presynaptic calcium dyshomeostasis, decreased levels of exocytosis, and a reduction in synaptic vesicle recruitment from the reserve pool during extended stimulation. These results support an active mitochondrial trafficking and fusion related QC process that supports presynaptic physiology. O_LIAnterograde trafficked mitochondria are larger and relatively more reduced than retrograde trafficked mitochondria C_LIO_LIAnterograde mitochondria fuse with and complement resident, stationary, axonal mitochondria C_LIO_LILoss of mitofusin 2 (MFN2) mediated mitochondrial fusion leads to alterations in the synaptic vesicle cycle and decreased reserve pool mobilization C_LI

neuroscience↗

bioRxiv2022

Multifunctional fluorophores for live-cell imaging and affinity capture of proteins

Enzyme-based self-labeling tags enable covalent attachment of synthetic molecules to proteins inside living cells. A frontier of this field is designing multifunctional ligands that contain both fluorophores and affinity tags or pharmacological agents and can still efficiently enter cells. Self-labeling tag ligands with short linkers can enter cells readily but often show less activity due to steric issues; ligands with long linkers can be more potent but show lower cell permeability. Here, we overcome this tug-of-war between efficacy and cell-permeability by devising a rational strategy for making cell permeable multifunctional ligands for labeling HaloTag fusions. We found that the lactone-zwitterion equilibrium sconstant (KL-Z) of rhodamines inversely correlates with their distribution coefficients (logD7.4), suggesting that ligands based on dyes exhibiting low KL-Z and high logD7.4 values, such as Si-rhodamines, would efficiently enter cells. We designed cell-permeable multifunctional HaloTag ligands with a biotin moiety to purify mitochondria or a JQ1 appendage to translocate BRD4 from euchromatin to the nucleolus or heterochromatin. We discovered that translocation of BRD4 to constitutive heterochromatin in cells expressing HaloTag-HP1a fusion proteins can lead to apparent increases in transcriptional activity. These new reagents enable affinity capture and translocation of intracellular proteins in living cells and the use of Si-rhodamines and other low KL- Z/high logD7.4 dye scaffolds will facilitate the design of new multifunctional chemical tools for biology. SIGNIFICANCE STATEMENTUnderstanding cellular processes requires tools to measure and manipulate proteins in living cells. Self- labeling tags, such as the HaloTag and SNAP-tag, enable modification of cellular proteins with synthetic molecules. Creating ligands for these systems that have more than one chemical motif remains challenging, however, due to competing demands between cell permeability and functionality. We discovered that multifunctional ligands based on Si-rhodamines efficiently entered cells and enabled affinity purification of mitochondria or translocation of nuclear proteins; the performance of these molecules could be verified by fluorescence microscopy. These compounds should be useful for a variety of biological experiments and our general framework will allow the design of other multifunctional ligands to study living systems.

cell biology↗

bioRxiv2022

Synaptic vesicle proteins are selectively delivered to axons in mammalian neurons

Neurotransmitter-filled synaptic vesicles (SV) mediate synaptic transmission and are a hallmark specialization in neuronal axons. Yet, how SV proteins are sorted to presynaptic nerve terminals remains the subject of debate. The leading models posit that these proteins are randomly trafficked throughout neurons and are selectively retained in presynaptic boutons. Here, we used the RUSH system, in conjunction with HaloTag labeling approaches, to study the egress of two distinct SV proteins from the soma of cultured neurons. In sharp contrast to the selective retention model, both proteins selectively and specifically entered axons and did not traffic through dendrites; only upon overexpression do SV proteins spillover into other compartments. Moreover, we observed that SV constituents were first delivered to the presynaptic plasma membrane before incorporation into SVs. These experiments reveal a new-found membrane trafficking pathway in classically polarized mammalian neurons and provide a glimpse at the first steps of SV biogenesis.

neuroscience↗

bioRxiv2021

Synaptotagmin 1 oligomerization via the juxtamembrane linker regulates spontaneous and evoked neurotransmitter release

Synaptotagmin-1 (syt1) is a Ca2+ sensor that regulates synaptic vesicle exocytosis. Cell-based experiments suggest that syt1 functions as a multimer, however biochemical and electron microscopy studies have yielded contradictory findings regarding putative self-association. Here, we performed dynamic light scattering on syt1 in solution, followed by electron microscopy, and used atomic force microscopy to study syt1 self-association on supported lipid bilayers under aqueous conditions. Ring-like multimers were clearly observed. Multimerization was enhanced by Ca2+ and required anionic phospholipids. Large ring-like structures ([~]180 nm) were reduced to smaller rings ([~]30 nm) upon neutralization of a cluster of juxtamembrane lysine residues; further substitution of residues in the second C2-domain completely abolished self-association. When expressed in neurons, syt1 mutants with graded reductions in self-association activity exhibited concomitant reductions in: a) clamping spontaneous release, and b) triggering and synchronizing evoked release. Thus, the juxtamembrane linker of syt1 plays a crucial role in exocytosis by mediating multimerization.

neuroscience↗

bioRxiv2021

Synaptotagmin 7 is enriched at the plasma membrane to promote vesicle docking and control synaptic plasticity

Synaptotagmin (SYT) 7 has emerged as key regulator of presynaptic function, but its localization and precise function in the synaptic vesicle cycle remain unclear. Here, we used iGluSnFR to optically and directly interrogate glutamate release, at the single bouton level, in SYT7 KO dissociated mouse hippocampal neurons. We analyzed asynchronous release, paired pulse facilitation, and synaptic vesicle replenishment, and found that SYT7 contributes to each of these processes to different degrees. Zap-and-freeze electron microscopy revealed that loss of SYT7 impairs the docking of synaptic vesicles after a stimulus and the recovery of depleted synaptic vesicles after a stimulus train. To execute these functions, SYT7 must be targeted to the plasma membrane via {gamma}-secretase-mediated cleavage of the amino terminus, followed by palmitoylation. The complex sorting itinerary of SYT7 endows this Ca2+-sensor with the ability to control crucial forms of synaptic function and plasticity. O_LISYT7 mediated asynchronous release, paired pulse facilitation, and synaptic vesicle replenishment was observed optically at individual hippocampal synapses C_LIO_LILocalization, trafficking, and stability of SYT7 is dependent on processing by {gamma}-secretase C_LIO_LIShort term plasticity defects arise in SYT7KOs due to decreased docking of synaptic vesicles after stimulation C_LIO_LISYT7 promotes paired-pulse facilitation and asynchronous release via distinct mechanisms C_LI

neuroscience↗