Biology subjects

Bewersdorf, J.

Publications and source records attributed to Bewersdorf, J..

3 recordsLinked to original sources

bioRxiv2017

Nano-scale size holes in ER sheets provide an alternative to tubules for highly-curved membranes

The endoplasmic reticulum (ER) is composed of interconnected membrane sheets and tubules. Super-resolution microscopy recently revealed densely packed, rapidly moving ER tubules, highlighting the importance of revisiting classical views of ER structure with high spatial resolution in living cells. Using live-cell Stimulated Emission Depletion (STED) microscopy, we show highly dynamic, subdiffraction-sized holes in ER sheets. Holes coexist with uniform sheet regions and are distinct from tubular ER structures. The curvature-stabilizing reticulon protein Rtn4 localizes to these holes and the ER luminal tether Climp63 controls their diameter and mobility. Analytical modeling demonstrates that holes in ER sheets can serve as reservoirs for curvature-stabilizing proteins to support ER tubule extension and retraction, thus providing an explanation for how the ER locally alters its morphology on fast time-scales.\n\nOne Sentence SummaryDynamic nano-scale sized holes are prominent features of ER sheets that serve as reservoirs for curvature-stabilizing proteins to support ER tubule extension and retraction.

cell biology↗

bioRxiv2017

Simultaneously measuring image features and resolution in live-cell STED images

Reliable interpretation and quantification of cellular features in fluorescence microscopy requires an accurate estimate of microscope resolution. This is typically obtained by measuring the image of a non-biological proxy for a point-like object, such as a fluorescent bead. While appropriate for confocal microscopy, bead-based measurements are problematic for Stimulated Emission Depletion (STED) and similar techniques where the resolution depends critically on the choice of fluorophore and acquisition parameters. We demonstrate that for a known geometry, e.g. tubules, the resolution can be accurately measured by fitting a model that accounts for both the Point Spread Function (PSF) and the fluorophore distribution. To address the problem of coupling between tubule diameter and PSF width, we developed a technique, Nested-loop Ensemble PSF (NEP) fitting. NEP fitting enables extraction of the size of cellular features and the PSF in fixed-cell and live-cell images without relying on beads or pre-calibration. We validate our technique using fixed microtubules and apply it to measure the diameter of endoplasmic reticulum tubules in live COS-7 cells. NEP fitting has been implemented as a plugin for the PYthon Microscopy Environment (PYME), a freely available and open source software.

cell biology↗

bioRxiv2017

Analysis Of Interphase Node Proteins In Fission Yeast By Quantitative And Super Resolution Fluorescence Microscopy

We used quantitative confocal microscopy and FPALM super resolution microscopy of live fission yeast to investigate the structures and assembly of two types of interphase nodes, multiprotein complexes associated with the plasma membrane that merge together and mature into the precursors of the cytokinetic contractile ring. During the long G2 phase of the cell cycle seven different interphase node proteins maintain constant concentrations as they accumulate in proportion to cell volume. During mitosis the total numbers of type 1 node proteins (cell cycle kinases Cdr1p, Cdr2p, Wee1p, and anillin Mid1p) are constant even when the nodes disassemble. Quantitative measurements provide strong evidence that both types of nodes have defined sizes and numbers of constituent proteins, as observed for cytokinesis nodes. Type 1 nodes assemble in two phases, a burst at the end of mitosis, followed by steady increase during interphase to double the initial number. Type 2 nodes containing Blt1p, Rho-GEF Gef2p, and kinesin Klp8p remain intact throughout the cell cycle and are constituents of the contractile ring. They are released from the contractile ring as it disassembles and then associate with type 1 nodes around the equator of the cell during interphase.\n\nHighlight summaryFPALM super resolution microscopy and quantitative confocal microscopy reveal that interphase nodes, the precursors to the fission yeast cytokinetic contractile ring, are discrete unitary structures with defined sizes and ratios of component proteins. Type 1 nodes disassemble during mitosis, but type 2 nodes remain intact throughout the cell cycle.

cell biology↗