Michael W. Gramlich

Fluorescence imaging of nanoscale domains in polymer blends using stochastic optical reconstruction microscopy (STORM)

High-resolution fluorescence techniques that provide spatial resolution below the diffraction limit are attractive new methods for structural characterization of nanostructured materials. For the first time, we apply the super-resolution technique of Stochastic Optical Reconstruction Microscopy (STORM), to characterize nanoscale structures within polymer blend films. The STORM technique involves temporally separating the fluorescence signals from individual labeled polymers, allowing their positions to be localized with high accuracy, yielding a high-resolution composite image of the material. Here, we describe the application of the technique to demixed blend films of polystyrene (PS) and poly(methyl methacrylate) (PMMA), and find that STORM provides comparable structural characteristics as those determined by Atomic Force Microscopy (AFM) and scanning electron microscopy (SEM), but with all of the advantages of a far-field optical technique.

Microtubule orientation and spacing within bundles is critical for long-range kinesin-1 motility.

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.

Single Molecule Investigation of Kinesin-1 Motility Using Engineered Microtubule Defects

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.

Actin/Myosin-V- and Activity-Dependent Inter-synaptic Vesicle Exchange in Central Neurons

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.

Activity-Dependence of Synaptic Vesicle Dynamics

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.