Zhang Y.,Biophysics Program |
Hoppe A.D.,University of Michigan |
Hoppe A.D.,South Dakota State University |
Swanson J.A.,Biophysics Program |
Swanson J.A.,University of Michigan
Proceedings of the National Academy of Sciences of the United States of America | Year: 2010
During Fcγ receptor (FcR)-mediated phagocytosis by macrophages, cytoplasmadvances over IgG-coated particles by the sequential ligation of FcR in plasma membranes. If FcR signaling was strictly autonomous, then the signals generated during phagocytosis should be proportional to the number of ligated receptors. By measuring FcRdependent responses to beads coated with various densities of IgG, this study identified nonlinear signaling that organizes an all or none response during particle ingestion. Phagocytosis of beads with IgG at low density either stalled after making small, actin-rich cups or proceeded to completion at the same rate as phagocytosis of highdensity IgGbeads. Signalsweremeasured by quantifying the recruitment of YFP-labeled probes to phagocytic cupmembranes. Although the magnitude of early signals correlated with IgG density, later signals showed an all or none response, which was regulated by the concentrations of 3′ phosphoinositides in phagocytic cup membranes. Thus, 3′ phosphoinositides, shown previously to be required for phagocytosis, function in a feedback regulatory mechanism affecting late but not early signals. This indicates a mechanism for the coordination of cell movements initiated by receptor signaling.
PubMed | Pennsylvania State University, Stanford University and Biophysics Program.
Type: Journal Article | Journal: Proceedings of the National Academy of Sciences of the United States of America | Year: 2014
Kinesin-1 is a dimeric motor protein, central to intracellular transport, that steps hand-over-hand toward the microtubule (MT) plus-end, hydrolyzing one ATP molecule per step. Its remarkable processivity is critical for ferrying cargo within the cell: over 100 successive steps are taken, on average, before dissociation from the MT. Despite considerable work, it is not understood which features coordinate, or gate, the mechanochemical cycles of the two motor heads. Here, we show that kinesin dissociation occurs subsequent to, or concomitant with, phosphate (P(i)) release following ATP hydrolysis. In optical trapping experiments, we found that increasing the steady-state population of the posthydrolysis ADP P(i) state (by adding free P(i)) nearly doubled the kinesin run length, whereas reducing either the ATP binding rate or hydrolysis rate had no effect. The data suggest that, during processive movement, tethered-head binding occurs subsequent to hydrolysis, rather than immediately after ATP binding, as commonly suggested. The structural change driving motility, thought to be neck linker docking, is therefore completed only upon hydrolysis, and not ATP binding. Our results offer additional insights into gating mechanisms and suggest revisions to prevailing models of the kinesin reaction cycle.
Sorelle E.D.,Biophysics Program |
Sorelle E.D.,Stanford University |
Liba O.,Stanford University |
Sen D.,Stanford University |
And 2 more authors.
Progress in Biomedical Optics and Imaging - Proceedings of SPIE | Year: 2017
Optical Coherence Tomography (OCT) is well-suited to study in vivo dynamics of blood circulation and lymphatic flow because of the technique's combination of rapid image acquisition, micron spatial resolution, and penetration depth in turbid tissues. However, OCT has been historically constrained by a dearth of contrast agents that are readily distinguished from the strong scattering intrinsic to biological tissues. In this study, we demonstrate large gold nanorods (LGNRs) as optimized contrast agents for OCT. LGNRs produce 32-fold greater backscattering than GNRs previously tested for contrast-enhanced OCT. Furthermore, LGNRs exhibit 110-fold stronger spectral signal than conventional GNRs when coupled with custom spectral detection algorithms. This signal enhancement enables picomolar OCT detection sensitivity in vivo and single-particle detection against optically-clear backgrounds. Moreover, the ability to synthesize LGNRs with tunable spectral peaks provides a viable platform for multiplexed imaging studies. To explore the advantages of LGNRs as OCT contrast agents, we implemented them for noninvasive 3D imaging of tumor blood supply and active lymphatic drainage in mice. Spectral detection of LGNRs enabled 100% improvement in imaging depth for detecting microvasculature (vessels ∼ 20 μm in diameter) in U87MG glioblastoma xenografts in mice pinnae. We also demonstrated our approach's ability to map the spatial dependence of lymph drainage and flow directionality within lymphatic capillaries. Using LGNRs with distinct spectra, we further identified the functional states of individual lymphatic valves in vivo. Thus, this approach provides a powerful new platform for functional imaging that may be extended for future molecular imaging studies with OCT. © 2017 SPIE.
Chang C.-W.,Physiology Graduate Training Program |
Chiang C.-W.,Biophysics Program |
Gaffaney J.D.,Howard Hughes Medical Institute |
Chapman E.R.,Biophysics Program |
And 4 more authors.
Journal of Biological Chemistry | Year: 2016
SNARE proteins catalyze many forms of biological membrane fusion, including Ca2+-triggered exocytosis. Although fusion mediated by SNAREs generally involves proteins anchored to each fusing membrane by a transmembrane domain (TMD), the role of TMDs remains unclear, and previous studies diverge on whether SNAREs can drive fusion without a TMD. This issue is important because it relates to the question of the structure and composition of the initial fusion pore, as well as the question of whether SNAREs mediate fusion solely by creating close proximity between two membranes versus a more active role in transmitting force to the membrane to deform and reorganize lipid bilayer structure. To test the role of membrane attachment, we generated four variants of the synaptic v-SNARE synaptobrevin- 2 (syb2) anchored to the membrane by lipid instead of protein. These constructs were tested for functional efficacy in three different systems as follows: Ca2+-triggered dense core vesicle exocytosis, spontaneous synaptic vesicle exocytosis, and Ca2+-synaptotagmin-enhanced SNARE-mediated liposome fusion. Lipid-anchoring motifs harboring one or two lipid acylation sites completely failed to support fusion in any of these assays. Only the lipid-anchoring motif from cysteine string protein-α, which harbors many lipid acylation sites, provided support for fusion but at levels well below that achieved with wild type syb2. Thus, lipid-anchored syb2 provides little or no support for exocytosis, and anchoring syb2 to a membrane by a TMD greatly improves its function. The low activity seen with syb2-cysteine string protein-α may reflect a slower alternative mode of SNARE-mediated membrane fusion. ©2016 by The American Society for Biochemistry and Molecular Biology, Inc.