Biology subjects

Fletcher, D. A.

Publications and source records attributed to Fletcher, D. A..

4 recordsLinked to original sources

bioRxiv2018

Low fidelity assembly of influenza A virus promotes escape from host cells

Influenza viruses inhabit a wide range of host environments using a limited repertoire of protein components. Unlike viruses with stereotyped shapes, influenza produces virions with significant morphological variability even within clonal populations. Whether this tendency to form pleiomorphic virions is coupled to compositional heterogeneity and whether it affects replicative fitness remains unclear. Here we address these questions by developing live strains of influenza A virus amenable to rapid compositional characterization through quantitative, site-specific labeling of viral proteins. Using these strains, we find that influenza A produces virions with broad variations in size and composition from even single infected cells. The virus leverages this phenotypic variability to survive environmental challenges including temperature changes and anti-virals. Complimenting genetic adaptations that act over larger populations and longer times, this low fidelity assembly of influenza A virus allows small populations to survive environments that fluctuate over individual replication cycles.

microbiology↗

bioRxiv2018

Claudin-4 Reconstituted in Unilamellar Vesicles is Sufficient to Form Tight Interfaces that Partition Membrane Proteins

Tight junctions have been hypothesized to act as molecular fences in the plasma membrane of epithelial cells, helping to form differentiated apical and basolateral domains. While this fence function is believed to arise from the interaction of four-pass transmembrane claudins, the complexity of tight junctions has made direct evidence of their role as a putative diffusion barrier difficult to obtain. Here we address this challenge by reconstituting claudin-4 into giant unilamellar vesicles using microfluidic jetting. We find that reconstituted claudin-4 is sufficient to form adhesive interfaces between unilamellar vesicles without accessory proteins present in vivo. By controlling the molecular composition of the inner and outer leaflets of jetted membranes, we show that claudin-4-mediated interfaces can drive partitioning of extracellular membrane proteins but not of inner or outer leaflet lipids. Our findings indicate that homotypic interactions of claudins and their small size can contribute to the polarization of epithelial cells.

cell biology↗

bioRxiv2018

Size-dependent segregation controls macrophage phagocytosis of antibody-opsonized targets

Macrophages protect the body from damage and disease by targeting antibody-opsonized cells for phagocytosis. Though antibodies can be raised against antigens with diverse structures, shapes, and sizes, it is unclear why some are more effective at triggering antibody-dependent phagocytosis than others. Here we quantitatively define an antigen height threshold that regulates phagocytosis of both engineered and cancer-specific antigens by macrophages. Using a reconstituted model of antibody-opsonized target cells, we find that phagocytosis is dramatically impaired for antigens that position antibodies >10 nm from the target surface. Increasing antigen height allows for co-localization of Fc receptors and the inhibitory phosphatase CD45 at the cell-cell interface, which reduces Fc receptor phosphorylation, and inhibits phagocytosis. Our work shows that close contact between macrophage and target cell is a requirement for efficient phagocytosis, suggesting that therapeutic antibodies should target short antigens in order to trigger Fc receptor activation through size-dependent physical segregation.

cell biology↗

bioRxiv2017

Spectrin is a mechanoresponsive protein shaping the architecture of intercellular invasion

Spectrin is a membrane skeletal protein best known for its structural role in maintaining cell shape and protecting cells from mechanical damage1-3. Here, we report that spectrin dynamically accumulates and dissolves at the fusogenic synapse, where an attacking fusion partner mechanically invades its receiving partner with actin-propelled protrusions to promote cell-cell fusion4-7. Using genetics, cell biology, biophysics and mathematical modeling, we demonstrate that unlike myosin II that responds to dilation deformation, spectrin exhibits a mechanosensitive accumulation in response to shear deformation, which is highly elevated at the fusogenic synapse. The accumulated spectrin forms an uneven network, which functions as a \"sieve\" to constrict the invasive fingerlike protrusions, thus putting the fusogenic synapse under high mechanical tension to promote cell membrane fusion. Taken together, our study has revealed a previously unrecognized function of spectrin as a dynamic mechanoresponsive protein that shapes the architecture of intercellular invasion. These findings have general implications for understanding spectrin function in other dynamic cellular processes beyond cell-cell fusion.

cell biology↗