Biology subjects

Bailey, E. M.

Publications and source records attributed to Bailey, E. M..

4 recordsLinked to original sources

bioRxiv2021

Anti-inflammatory therapy protects spiral ganglion neurons after aminoglycoside antibiotic-induced hair cell loss

AO_SCPLOWBSTRACTC_SCPLOWSpiral ganglion neurons (SGNs) relay auditory information from cochlear hair cells to the central nervous system. After hair cells are destroyed by aminoglycoside antibiotics, SGNs gradually die. However, the reasons for this cochlear neurodegeneration are unclear. We used microarray gene expression profiling to assess transcriptomic changes in the spiral ganglia of kanamycin-deafened and age-matched control rats and found that many of the genes upregulated after deafening are associated with immune/inflammatory responses. In support of this, we observed increased numbers of macrophages in the spiral ganglion of deafened rats. We also found, via CD68 immunoreactivity, an increase in activated macrophages after deafening. An increase in CD68-associated nuclei was observed by postnatal day 23, a time before significant SGN degeneration is observed. Finally, we show that the immunosuppressive drugs dexamethasone and ibuprofen, as well as the NAD salvage pathway activator P7C3, provide at least some neuroprotection post-deafening. Ibuprofen and dexamethasone also decreased the degree of macrophage activation. These results suggest that activated macrophages specifically, and perhaps a more general neuroinflammatory response, are actively contributing to SGN degeneration after hair cell loss.

neuroscience↗

bioRxiv2021

IgG surface mobility promotes antibody-dependent cellular phagocytosis by Syk and Arp2/3 mediated reorganization of Fcγ receptors in macrophages.

By visualizing the movements of Rituximab during Antibody dependent cellular phagocytosis (ADCP) of B lymphoma cells by macrophages, we found that Fc{gamma} receptors (Fc{gamma}R) on the macrophage surface microcluster, recruit Syk and undergro large-scale reorganization at the phagocytic synapse prior to and during engulfment of the target cell. Given these dramatic rearrangements, we analyzed how the surface mobility of Rituximab contributes to Fc{gamma}R signal amplification and ADCP efficiency. Depolymerization of the target cell actin cytoskeleton resulted in free diffusion of Rituximab docked to CD20, enhanced microcluster reorganization, Syk recruitment and ADCP. Conversely, immobilization of Rituximab by chemical fixation impaired microcluster formation and diminished Syk recruitment and ADCP. In macrophages lacking Syk, Rituximab accumulated at the base of the phagosome and were trogocytosed, consistent with Syk kinase activity being necessary to trigger redistribution of Rituximab-Fc{gamma}R during engulfment and to prevent antigenic modulation of the target. Total internal reflection fluorescence analysis of Fc{gamma}R-IgG on fluid supported lipid bilayers revealed a membrane topography displaying inward reaching leading edges and protruding contact sites reminiscent of podosomes. This topography was distinct from the closely apposed macrophage/target membranes observed during engagement of IgG displayed on immobile supported lipid bilayers. The organization of this contact, pseudopod extension and the rearrangement of microclusters depended critically on Arp 2/3. Thus, Syk and Arp2/3 coordinate actin rearrangements and Fc{gamma}R-IgG complexes that were of previously unrecognized complexity for the clearance of cells displaying surface-mobile antigens. Significance StatementADCP is an important effector mechanism for the removal of malignant, immunologically aberrant, and infected cells during treatment with therapeutic antibodies or adaptive immune responses. Most transmembrane protein antigens are mobile with transient confinement from the actin of the target cell. This work demonstrates that macrophage forces overcome these confinements to rearrange Fc{gamma}R-IgG-antigen complexes before and during ADCP. Thus, new paradigms are needed as ADCP has largely been studied using model target particles that display immobile antigens. Moreover, we found that the mobility of the therapeutic antibody, Rituximab, on the surface of B lymphoma cells foretells ADCP efficacy, with lower densities of IgG mediating ADCP on increasingly mobile antigens.

immunology↗

bioRxiv2021

Sterols lower energetic barriers of membrane bending and fission necessary for efficient clathrin mediated endocytosis

As the principal internalization mechanism in mammalian cells, clathrin-mediated endocytosis (CME) is critical for cellular signal transduction, receptor recycling, and membrane homeostasis. Acute depletion of cholesterol disrupts CME, motivating analysis of CME dynamics in the context of disrupted cholesterol synthesis, sterol specificity, mechanisms involved, and relevance to disease pathology. Using genome-edited cell lines, we demonstrate that inhibition of post-squalene cholesterol biosynthesis as observed in inborn errors of cholesterol metabolism, results in striking immobilization of CME and impaired transferrin uptake. Imaging of membrane bending dynamics and CME pit ultrastructure revealed prolonged clathrin pit lifetimes and accumulation of shallow clathrin-coated structures that scaled with diminishing sterol abundance. Moreover, fibroblasts derived from Smith-Lemli-Opitz syndrome subjects displayed reduced CME function. We conclude that sterols lower the energetic costs of membrane bending during pit formation and vesicular scission during CME and suggest reduced CME contributes to cellular phenotypes observed within disorders of cholesterol metabolism.

cell biology↗

bioRxiv2017

Membrane bending begins at any stage of clathrin-coat assembly and defines endocytic dynamics

Summary ParagraphClathrin-mediated endocytosis internalizes membrane from the cell surface by reshaping flat regions of membrane into spherical vesicles(1, 2). The relationship between membrane bending and clathrin coatomer assembly has been inferred from electron microscopy and structural biology, without directly visualization of membrane bending dynamics (3-6). This has resulted in two distinct and opposing models for how clathrin bends membrane (7-10). Here, polarized Total Internal Reflection Fluorescence microscopy was improved and combined with electron microscopy, atomic force microscopy, and super-resolution imaging to measure membrane bending during endogenous clathrin and dynamin assembly in living cells. Surprisingly, and not predicted by either model, the timing of membrane bending was variable relative to clathrin assembly. Approximately half of the time, membrane bending occurs at the start of clathrin assembly, in the other half, the onset of membrane bending lags clathrin arrival, and occasionally completely assembled flat clathrin transitions into a pit. Importantly, once the membrane bends, the process proceeds to scission with similar timing. We conclude that the pathway of coatomer formation is versatile and can bend the membrane during or after the assembly of the clathrin lattice. These results highlight the heterogeneity in this fundamental biological process, and provide a more complete nanoscale view of membrane bending dynamics during endocytosis.

cell biology↗