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1.
Sci Rep ; 14(1): 11912, 2024 05 24.
Article in English | MEDLINE | ID: mdl-38789439

ABSTRACT

The objective of this study is to determine characteristics of patients with myofascial pain syndrome (MPS) of the low back and the degree to which the low back pain in the patients examined can be attributed to MPS. Twenty-five subjects with myofascial trigger point(s) [MTrP(s)] on the low back participated in this cross-sectional study. The location, number, and type of selected MTrPs were identified by palpation and verified by ultrasound. Pain pressure threshold, physical function, and other self-reported outcomes were measured. Significant differences were found in Group 1 (Active), 2 (Latent), 3 (Atypical, no twitching but with spontaneous pain), and 4 (Atypical, no twitching and no spontaneous pain) of participants in the number of MTrPs, current pain, and worst pain in the past 24 h (p = .001-.01). There were interaction effects between spontaneous pain and twitching response on reports of physical function, current pain, and worst pain (p = .002-.04). Participants in Group 3 reported lower levels of physical function, and higher levels of current pain and worst pain compared to those in Group 4. Participants in Group 1 and 2 had similar levels of physical function, current pain, and worst pain. The number of MTrPs is most closely associated with the level of pain. Spontaneous pain report seems to be a decisive factor associated with poor physical function; however, twitching response is not.


Subject(s)
Low Back Pain , Myofascial Pain Syndromes , Humans , Female , Male , Myofascial Pain Syndromes/physiopathology , Adult , Cross-Sectional Studies , Low Back Pain/physiopathology , Middle Aged , Trigger Points/physiopathology , Pain Measurement , Pain Threshold , Ultrasonography
2.
Front Cell Dev Biol ; 11: 1286915, 2023.
Article in English | MEDLINE | ID: mdl-38020880

ABSTRACT

Presynapses locally recycle synaptic vesicles to efficiently communicate information. During use and recycling, proteins on the surface of synaptic vesicles break down and become less efficient. In order to maintain efficient presynaptic function and accommodate protein breakdown, new proteins are regularly produced in the soma and trafficked to presynaptic locations where they replace older protein-carrying vesicles. Maintaining a balance of new proteins and older proteins is thus essential for presynaptic maintenance and plasticity. While protein production and turnover have been extensively studied, it is still unclear how older synaptic vesicles are trafficked back to the soma for recycling in order to maintain balance. In the present study, we use a combination of fluorescence microscopy, hippocampal cell cultures, and computational analyses to determine the mechanisms that mediate older synaptic vesicle trafficking back to the soma. We show that synaptic vesicles, which have recently undergone exocytosis, can differentially utilize either the microtubule or the actin cytoskeleton networks. We show that axonally trafficked vesicles traveling with higher speeds utilize the microtubule network and are less likely to be captured by presynapses, while slower vesicles utilize the actin network and are more likely to be captured by presynapses. We also show that retrograde-driven vesicles are less likely to be captured by a neighboring presynapse than anterograde-driven vesicles. We show that the loss of synaptic vesicle with bound molecular motor myosin V is the mechanism that differentiates whether vesicles will utilize the microtubule or actin networks. Finally, we present a theoretical framework of how our experimentally observed retrograde vesicle trafficking bias maintains the balance with previously observed rates of new vesicle trafficking from the soma.

3.
Dev Cell ; 58(19): 1847-1863.e12, 2023 10 09.
Article in English | MEDLINE | ID: mdl-37751746

ABSTRACT

An actin-spectrin lattice, the membrane periodic skeleton (MPS), protects axons from breakage. MPS integrity relies on spectrin delivery via slow axonal transport, a process that remains poorly understood. We designed a probe to visualize endogenous spectrin dynamics at single-axon resolution in vivo. Surprisingly, spectrin transport is bimodal, comprising fast runs and movements that are 100-fold slower than previously reported. Modeling and genetic analysis suggest that the two rates are independent, yet both require kinesin-1 and the coiled-coil proteins UNC-76/FEZ1 and UNC-69/SCOC, which we identify as spectrin-kinesin adaptors. Knockdown of either protein led to disrupted spectrin motility and reduced distal MPS, and UNC-76 overexpression instructed excessive transport of spectrin. Artificially linking spectrin to kinesin-1 drove robust motility but inefficient MPS assembly, whereas impairing MPS assembly led to excessive spectrin transport, suggesting a balance between transport and assembly. These results provide insight into slow axonal transport and MPS integrity.


Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans , Spectrin , Animals , Axonal Transport , Axons/metabolism , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Kinesins/metabolism , Spectrin/metabolism
4.
Front Synaptic Neurosci ; 14: 925546, 2022.
Article in English | MEDLINE | ID: mdl-35989711

ABSTRACT

The molecular pathways that contribute to the onset of symptoms in tauopathy models, including Alzheimer's disease (AD), are difficult to distinguish because multiple changes can happen simultaneously at different stages of disease progression. Understanding early synaptic alterations and their supporting molecular pathways is essential to develop better pharmacological targets to treat AD. Here, we focus on an early onset rTg(TauP301L )4510 tauopathy mouse model that exhibits hyperexcitability in hippocampal neurons of adult mice that is correlated with presynaptic changes and increased extracellular glutamate levels. However, it is not clear if increased extracellular glutamate is caused by presynaptic changes alone, or if presynaptic changes are a contributing factor among other factors. To determine whether pathogenic tau alters presynaptic function and glutamate release, we studied cultured hippocampal neurons at 14-18 days in vitro (DIV) from animals of both sexes to measure presynaptic changes in tauP301L positive mice. We observed that presynaptic vesicles exhibit increased vesicular glutamate transporter 1 (VGlut1) using immunohistochemistry of fixed cells and an established pH-sensitive green fluorescent protein approach. We show that tauP301L positive neurons exhibit a 40% increase in VGlut1 per vesicle compared to tauP301L negative littermates. Further, we use the extracellular glutamate reporter iGluSnFR to show that increased VGlut1 per vesicle directly translates into a 40% increase in extracellular glutamate. Together, these results show that increased extracellular glutamate levels observed in tauP301L mice are not caused by increased vesicle exocytosis probability but rather are directly related to increased VGlut1 transporters per synaptic vesicle.

5.
Front Synaptic Neurosci ; 13: 650334, 2021.
Article in English | MEDLINE | ID: mdl-33935678

ABSTRACT

Synaptic active zone (AZ) contains multiple specialized release sites for vesicle fusion. The utilization of release sites is regulated to determine spatiotemporal organization of the two main forms of synchronous release, uni-vesicular (UVR) and multi-vesicular (MVR). We previously found that the vesicle-associated molecular motor myosin V regulates temporal utilization of release sites by controlling vesicle anchoring at release sites in an activity-dependent manner. Here we show that acute inhibition of myosin V shifts preferential location of vesicle docking away from AZ center toward periphery, and results in a corresponding spatial shift in utilization of release sites during UVR. Similarly, inhibition of myosin V also reduces preferential utilization of central release sites during MVR, leading to more spatially distributed and temporally uniform MVR that occurs farther away from the AZ center. Using a modeling approach, we provide a conceptual framework that unites spatial and temporal functions of myosin V in vesicle release by controlling the gradient of release site release probability across the AZ, which in turn determines the spatiotemporal organization of both UVR and MVR. Thus myosin V regulates both temporal and spatial utilization of release sites during two main forms of synchronous release.

6.
Trends Neurosci ; 42(6): 425-437, 2019 06.
Article in English | MEDLINE | ID: mdl-31176424

ABSTRACT

Presynaptic boutons support neurotransmitter release with nanoscale precision at sub-millisecond timescales. Studies over the past two decades have revealed a rich tapestry of molecular players governing synaptic vesicle fusion at highly specialized release sites in the active zone (AZ). However, the spatiotemporal organization of release at active synapses remains elusive, in part owing to the extremely small size of the AZ and the limited resolution of conventional approaches. Recent advances in fluorescence nanoscopy have revolutionized direct investigation of presynaptic release organization and dynamics. We discuss here recent nanoscopy-based studies of the molecular architecture, the spatial organization and dynamic regulation of release sites, and the mechanisms of release site replenishment. These findings have uncovered previously unknown levels of structural and functional organization at central synapses, with important implications for synaptic transmission and plasticity.


Subject(s)
Presynaptic Terminals/physiology , Presynaptic Terminals/ultrastructure , Synaptic Transmission/physiology , Synaptic Vesicles/physiology , Synaptic Vesicles/ultrastructure , Animals , Exocytosis/physiology , Humans , Nanotechnology/methods
7.
Elife ; 72018 10 15.
Article in English | MEDLINE | ID: mdl-30320552

ABSTRACT

Synaptic vesicle fusion occurs at specialized release sites at the active zone. How refilling of release sites with new vesicles is regulated in central synapses remains poorly understood. Using nanoscale-resolution detection of individual release events in rat hippocampal synapses we found that inhibition of myosin V, the predominant vesicle-associated motor, strongly reduced refilling of the release sites during repetitive stimulation. Single-vesicle tracking revealed that recycling vesicles continuously shuttle between a plasma membrane pool and an inner pool. Vesicle retention at the membrane pool was regulated by neural activity in a myosin V dependent manner. Ultrastructural measurements of vesicle occupancy at the plasma membrane together with analyses of single-vesicle trajectories during vesicle shuttling between the pools suggest that myosin V acts as a vesicle tether at the plasma membrane, rather than a motor transporting vesicles to the release sites, or directly regulating vesicle exocytosis.


Subject(s)
Cell Membrane/metabolism , Myosin Type V/metabolism , Neurotransmitter Agents/metabolism , Synapses/metabolism , Animals , Hippocampus/metabolism , Models, Biological , Presynaptic Terminals/metabolism , Presynaptic Terminals/ultrastructure , Protein Transport , Rats , Synaptic Vesicles/metabolism
8.
J Neurosci ; 37(44): 10597-10610, 2017 11 01.
Article in English | MEDLINE | ID: mdl-28954868

ABSTRACT

The proper function of synapses relies on efficient recycling of synaptic vesicles. The small size of synaptic boutons has hampered efforts to define the dynamical states of vesicles during recycling. Moreover, whether vesicle motion during recycling is regulated by neural activity remains largely unknown. We combined nanoscale-resolution tracking of individual synaptic vesicles in cultured hippocampal neurons from rats of both sexes with advanced motion analyses to demonstrate that the majority of recently endocytosed vesicles undergo sequences of transient dynamical states including epochs of directed, diffusional, and stalled motion. We observed that vesicle motion is modulated in an activity-dependent manner, with dynamical changes apparent in ∼20% of observed boutons. Within this subpopulation of boutons, 35% of observed vesicles exhibited acceleration and 65% exhibited deceleration, accompanied by corresponding changes in directed motion. Individual vesicles observed in the remaining ∼80% of boutons did not exhibit apparent dynamical changes in response to stimulation. More quantitative transient motion analyses revealed that the overall reduction of vesicle mobility, and specifically of the directed motion component, is the predominant activity-evoked change across the entire bouton population. Activity-dependent modulation of vesicle mobility may represent an important mechanism controlling vesicle availability and neurotransmitter release.SIGNIFICANCE STATEMENT Mechanisms governing synaptic vesicle dynamics during recycling remain poorly understood. Using nanoscale resolution tracking of individual synaptic vesicles in hippocampal synapses and advanced motion analysis tools we demonstrate that synaptic vesicles undergo complex sets of dynamical states that include epochs of directed, diffusive, and stalled motion. Most importantly, our analyses revealed that vesicle motion is modulated in an activity-dependent manner apparent as the reduction in overall vesicle mobility in response to stimulation. These results define the vesicle dynamical states during recycling and reveal their activity-dependent modulation. Our study thus provides fundamental new insights into the principles governing synaptic function.


Subject(s)
Endocytosis/physiology , Hippocampus/physiology , Neurons/physiology , Synaptic Vesicles/physiology , Animals , Animals, Newborn , Cells, Cultured , Female , Hippocampus/cytology , Male , Presynaptic Terminals/physiology , Rats , Synapses/physiology
9.
Cell Rep ; 18(9): 2096-2104, 2017 02 28.
Article in English | MEDLINE | ID: mdl-28249156

ABSTRACT

Vesicle sharing between synaptic boutons is an important component of the recycling process that synapses employ to maintain vesicle pools. However, the mechanisms supporting and regulating vesicle transport during the inter-synaptic exchange remain poorly understood. Using nanometer-resolution tracking of individual synaptic vesicles and advanced computational algorithms, we find that long-distance axonal transport of synaptic vesicles between hippocampal boutons is partially mediated by the actin network, with myosin V as the primary actin-dependent motor that drives this vesicle transport. Furthermore, we find that vesicle exit from the synapse to the axon and long-distance vesicle transport are both rapidly and dynamically regulated by activity. We corroborated these findings with two complementary modeling approaches of vesicle exit, which closely reproduced experimental observations. These findings uncover the roles of actin and myosin V in supporting the inter-synaptic vesicle exchange and reveal that this process is dynamically modulated in an activity-dependent manner.


Subject(s)
Actins/metabolism , Myosin Type V/metabolism , Neurons/metabolism , Neurons/physiology , Synapses/metabolism , Synaptic Vesicles/metabolism , Synaptic Vesicles/physiology , Animals , Axonal Transport/physiology , Axons/metabolism , Axons/physiology , Cells, Cultured , Hippocampus/metabolism , Hippocampus/physiology , Presynaptic Terminals/metabolism , Presynaptic Terminals/physiology , Rats , Synapses/physiology , Synaptic Transmission/physiology
10.
Sci Rep ; 7: 44290, 2017 03 13.
Article in English | MEDLINE | ID: mdl-28287156

ABSTRACT

The structure of the microtubule is tightly regulated in cells via a number of microtubule associated proteins and enzymes. Microtubules accumulate structural defects during polymerization, and defect size can further increase under mechanical stresses. Intriguingly, microtubule defects have been shown to be targeted for removal via severing enzymes or self-repair. The cell's control in defect removal suggests that defects can impact microtubule-based processes, including molecular motor-based intracellular transport. We previously demonstrated that microtubule defects influence cargo transport by multiple kinesin motors. However, mechanistic investigations of the observed effects remained challenging, since defects occur randomly during polymerization and are not directly observable in current motility assays. To overcome this challenge, we used end-to-end annealing to generate defects that are directly observable using standard epi-fluorescence microscopy. We demonstrate that the annealed sites recapitulate the effects of polymerization-derived defects on multiple-motor transport, and thus represent a simple and appropriate model for naturally-occurring defects. We found that single kinesins undergo premature dissociation, but not preferential pausing, at the annealed sites. Our findings provide the first mechanistic insight to how defects impact kinesin-based transport. Preferential dissociation on the single-molecule level has the potential to impair cargo delivery at locations of microtubule defect sites in vivo.


Subject(s)
Computer Simulation , Kinesins/metabolism , Microtubules/metabolism , Models, Theoretical , Animals , Biological Transport , Brain/metabolism , Cattle , Kinesins/chemistry , Microscopy, Fluorescence/methods , Microtubules/chemistry , Polymerization , Swine , Tubulin/chemistry , Tubulin/metabolism
11.
Cytoskeleton (Hoboken) ; 71(11): 595-610, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25382100

ABSTRACT

Cells rely on active transport to quickly organize cellular cargo. How cells regulate transport is not fully understood. One proposed mechanism is that motor activity could be altered through the architecture of the cytoskeleton. This mechanism is supported by the fact that the cytoskeletal network is tightly regulated in cells and filament polarity within networks dictates motor directionality. For instance, axons contain bundles of parallel microtubules and all cargos with the same motor species will move in the same direction. It is not clear how other types of networks, such as antiparallel bundles in dendrites, can regulate motor transport. To understand how the organization of microtubules within bundles can regulate transport, we studied kinesin-1 motility on three bundle types: random-polarity bundles that are close-packed, parallel polarity bundles, and antiparallel polarity bundles that are spaced apart. We find that close-packed bundles inhibit motor motion, while parallel arrays support unidirectional motion. Spacing the microtubules with microtubule-associated proteins enhances run lengths. Our results indicate that microtubule bundle architecture dictates the motion of single motors and could have effects on cargo transport. © 2014 Wiley Periodicals, Inc.


Subject(s)
Kinesins/metabolism , Microtubule-Associated Proteins/metabolism , Molecular Motor Proteins/metabolism , Biological Transport , Cell Movement
12.
Integr Biol (Camb) ; 5(11): 1324-33, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24061278

ABSTRACT

Microtubules are essential protein filaments required to organize and rearrange the interior of the cell. They must be stiff with mechanical integrity to support the structure of the cell. Yet, they must also be dynamic to enable rearrangements of the cell during cell division and development. This dynamic nature is inherent to microtubules and comes about through the hydrolysis of chemical energy stored in guanosine triphosphate (GTP). Dynamic instability has been studied with a number of microscopy techniques both in cells and in reconstituted systems. In this article, we review the techniques used to examine microtubule dynamic instability and highlight future avenues and still open questions about this vital and fascinating activity.


Subject(s)
Microscopy/methods , Microtubules/physiology , Animals , Cell Division , Cytoskeleton/metabolism , Guanosine Triphosphate/chemistry , Humans , Microscopy, Confocal/methods , Microscopy, Fluorescence/methods , Microtubules/metabolism , Spindle Apparatus/metabolism , Tubulin/chemistry
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