Biology subjects

Manley, S.

Publications and source records attributed to Manley, S..

27 records · Page 2Linked to original sources

bioRxiv2020

Homogeneous multifocal excitation for high-throughput super-resolution imaging

Super-resolution microscopies, which allow features below the diffraction limit to be resolved, have become an established tool in biological research. However, imaging throughput remains a major bottleneck in using them for quantitative biology, which requires large datasets to overcome the noise of the imaging itself and to capture the variability inherent to biological processes. Here, we develop a multi-focal flat illumination for field independent imaging (mfFIFI) module, and integrate it into an instant structured illumination microscope (iSIM). Our instrument extends the field of view (FOV) to >100x100 {micro}m2 without compromising image quality, and maintains high-speed (100 Hz), multi-color, volumetric imaging at double the diffraction-limited resolution. We further extend the effective FOV by stitching multiple adjacent images together to perform fast live-cell super-resolution imaging of dozens of cells. Finally, we combine our flat-fielded iSIM setup with ultrastructure expansion microscopy (U-ExM) to collect 3D images of hundreds of centrioles in human cells, as well as of thousands of purified Chlamydomonas reinhardtii centrioles per hour at an effective resolution of [~]35 nm. We apply classification and particle averaging to these large datasets, allowing us to map the 3D organization of post-translational modifications of centriolar microtubules, revealing differences in their coverage and positioning.

biophysics↗

bioRxiv2019

Mitochondrial RNA granules are fluid condensates, positioned by membrane dynamics

Mitochondria contain the genetic information and expression machinery to produce proteins essential for cellular respiration. Within the mitochondrial matrix, newly synthesized RNA, RNA processing proteins, and mitoribosome assembly factors are known to form punctate subcompartments referred to as mitochondrial RNA granules (MRGs) 1-3. Despite their proposed role in regulating gene expression, little is known about the structural and dynamic properties of MRGs. We investigated the organization of MRGs using fluorescence super-resolution localization microscopy and correlative electron microscopy techniques, obtaining ultrastructural details of their internal architecture. We find that MRGs are organized into nanoscale RNA cores surrounded by a protein shell. Using live-cell super-resolution structured illumination microscopy and photobleaching perturbations, we reveal that MRGs undergo fusion and rapidly exchange components, consistent with liquid-liquid phase separation (LLPS). Furthermore, MRGs associate with the inner mitochondrial membrane and their fusion coincides with membrane remodeling. Inhibition of mitochondrial fission leads to an aberrant distribution of MRGs into concentrated pockets, where they remain as distinct individual units despite their close apposition. Together, our results reveal a role for LLPS in concentrating RNA and its processing proteins into MRGs, which are positioned along mitochondria by membrane dynamics.

biophysics↗

bioRxiv2019

Multi-phosphorylation reaction and clustering tune Pom1 gradient mid-cell levels according to cell size

Protein concentration gradients convey information at a distance from the source to both pattern developing organisms and organize single cells. In the rod-shaped cells of Schizosaccharomyces pombe, the DYRK-family kinase Pom1 forms concentration gradients with maxima at the cell poles. Pom1 controls the timing of mitotic entry by inhibiting the SAD-family kinase Cdr2, which forms stable membrane-associated nodes at mid-cell. Pom1 gradients rely on membrane association regulated by a phosphorylation-dephosphorylation cycle and lateral diffusion modulated by clustering. Whether the graded pattern directly alters Pom1 medial levels has been controversial. Here, using a combination of quantitative imaging approaches, including single particle tracking PALM and TIRF microscopy, we find that individual Pom1 molecules do not bind the membrane long enough to diffuse from cell pole to cell middle. Instead we propose they exchange within longer-lived clusters that form the functional gradient units. By creating an allelic series progressively blocking auto-phosphorylation, we show that multi-phosphorylation shapes and buffers the gradient to control the cortical mid-cell Pom1 levels, which represent the critical pool regulating Cdr2. Specific imaging of this cortical pool by TIRF microscopy demonstrates that more Pom1 overlaps with Cdr2 nodes in short than long cells, consistent with Pom1 inhibition of Cdr2 decreasing with cell growth. We conclude that Pom1 gradients modulate Pom1 mid-cell levels according to cell size.

cell biology↗

bioRxiv2018

Design Principles for Autonomous Illumination Control in Localization Microscopy

Super-resolution fluorescence microscopy improves spatial resolution, but this comes at a loss of image throughput and presents unique challenges in identifying optimal acquisition parameters. Microscope automation routines can offset these drawbacks, but thus far have required user inputs that presume a priori knowledge about the sample. Here, we develop a flexible illumination control system for localization microscopy comprised of two interacting components that require no sample-specific inputs: a self-tuning controller and a deep learning molecule density estimator that is accurate over an extended range. This system obviates the need to fine-tune parameters and demonstrates the design of modular illumination control for localization microscopy.

biophysics↗

bioRxiv2018

Constriction rate modulation can drive cell size control and homeostasis in C. crescentus

Rod-shaped bacteria typically grow first via sporadic and dispersed elongation along their lateral walls, then via a combination of zonal elongation and constriction at the division site to form the poles of daughter cells. Although constriction comprises up to half of the cell cycle, its impact on cell size control and homeostasis has rarely been considered. To reveal the roles of cell elongation and constriction in bacterial size regulation during cell division, we captured the shape dynamics of Caulobacter crescentus with time-lapse structured illumination microscopy and used molecular markers as cell-cycle landmarks. We perturbed constriction rate using a hyperconstriction mutant or fosfomycin inhibition. We report that constriction rate contributes to both size control and homeostasis, by determining elongation during constriction, and by compensating for variation in pre-constriction elongation on a single-cell basis.

biophysics↗

bioRxiv2018

Influenza A viruses use multivalent sialic acid clusters for cell binding and receptor activation

Influenza A virus (IAV) binds its host cell using the major viral surface protein hemagglutinin (HA). HA recognizes sialic acid, a plasma membrane glycan that functions as the specific primary attachment factor (AF). Since sialic acid alone cannot fulfill a signaling function, the virus needs to activate downstream factors to trigger endocytic uptake. Recently, the epidermal growth factor receptor (EGFR), a member of the receptor-tyrosine kinase family, was shown to be activated by and transmit IAV entry signals. However, how IAV engages and activates EGFR remains largely unclear.\n\nWe used multicolor super-resolution microscopy to study the lateral organization of both IAV attachment factors and its functional receptor at the scale of the IAV particle. Intriguingly, quantitative cluster analysis revealed that AF and EGFR are organized in partially overlapping submicrometer clusters in the apical plasma membrane of A549 cells. Within AF domains, which are distinct from microvilli, the local AF concentration, a parameter that directly influences virus-cell binding, reaches on average 10-fold the background concentration and tends to increase towards the cluster center, thereby representing a multivalent virus-binding platform. Using our experimentally measured cluster characteristics, we simulated virus diffusion on a membrane, revealing that the distinct mobility pattern of IAVs is dominated by the local AF concentration, consistent with live cell single-virus tracking data. In contrast to AF, EGFR resides in clusters of rather low molecular density. Virus binding activates EGFR, but interestingly, this process occurs without a major lateral EGFR redistribution, instead relying on activation of pre-formed clusters, which we show are long-lived.\n\nTaken together, our results provide a quantitative understanding of the initial steps of influenza virus infection. Co-clustering of AF and EGFR permit a cooperative effect of binding and signaling at specific platforms, and thus we relate their spatial organization to their functional role during virus-cell binding and receptor activation.\n\nAuthor SummaryThe plasma membrane is the major interface between a cell and its environment. It is a complex and dynamic organelle that needs to protect as a barrier but also process subtle signals into and out of the cell. For IAV, an enveloped virus, it represents a major obstacle that it needs to overcome during infection as well as the site for the assembly of progeny virus particles. However, the organisation of the plasma membrane in particular the sites of virus interaction at the scale of an infecting particle (length scales < 100 nm) remains largely unknown.\n\nSialic acids serve as IAV attachment factors but are not able to transmit signals across the plasma membrane. Receptor tyrosine kinases were identified to be activated upon virus binding and serve as functional receptor. How IAV engages and activates its functional receptors still remains speculative. Here we use super resolution microscopy to study the lateral organization as well as the functional relationship of plasma membrane-bound molecules involved in IAV infection. We find that molecules are organized in submicrometer nanodomains and, in combination with virus diffusion simulations, present a mechanistic view for how IAV first engages with AFs in the plasma membrane to then engage and trigger entry-associated membrane receptors.

microbiology↗

bioRxiv2018

Membrane bending energy and tension govern mitochondrial division

During mitochondrial fission, key molecular and cellular factors assemble on the outer mitochondrial membrane, where they coordinate to generate constriction. Constriction sites can eventually divide, or reverse upon disassembly of the machinery. However, a role for membrane tension in mitochondrial fission, although speculated, has remained undefined. We captured the dynamics of constricting mitochondria in mammalian cells using live-cell structured illumination microscopy (SIM). By analyzing the diameters of tubules that emerge from mitochondria and implementing a fluorescence lifetime-based mitochondrial membrane tension sensor, we discovered that mitochondria are indeed under tension. Under perturbations that reduce mitochondrial tension, constrictions initiate at the same rate, but are less likely to divide. We propose a model based on our estimates of mitochondrial membrane tension and bending energy in living cells which accounts for the observed probability distribution for mitochondrial constrictions to divide.

biophysics↗

bioRxiv2018

Multicolor single particle reconstruction of protein complexes

Single-particle reconstruction (SPR) from electron microscopy images is widely used in structural biology, but lacks direct information on protein identity. To address this limitation, we developed a computational and analytical framework that reconstructs and co-aligns multiple proteins from 2D super-resolution fluorescence images. We demonstrate our method by generating multi-color 3D reconstructions of several proteins within the human centriole and procentriole, revealing their relative locations, dimensions and orientations.

cell biology↗

bioRxiv2018

Single-molecule dynamics and genome-wide transcriptomics reveal that NF-kB (p65)-DNA binding times can be decoupled from transcriptional activation

Transcription factors (TFs) regulate gene expression in both prokaryotes and eukaryotes by recognizing and binding to specific DNA promoter sequences. In higher eukaryotes, it remains unclear how the duration of TF binding to DNA relates to downstream transcriptional output. Here, we address this question for the transcriptional activator NF-{kappa}B (p65), by live-cell single molecule imaging of TF-DNA binding kinetics and genome-wide quantification of p65-mediated transcription. We used mutants of p65, perturbing either the DNA binding domain (DBD) or the protein-protein transactivation domain (TAD). We found that p65-DNA binding time was predominantly determined by its DBD and directly correlated with its transcriptional output as long as the TAD is intact. Surprisingly, mutation or deletion of the TAD did not modify p65-DNA binding stability, suggesting that the p65 TAD generally contributes neither to the assembly of an \"enhanceosome,\" nor to the active removal of p65 from putative specific binding sites. However, TAD removal did reduce p65-mediated transcriptional activation, indicating that protein-protein interactions act to translate the long-lived p65-DNA binding into productive transcription.\n\nAuthor SummaryTo control transcription of a certain gene or a group of genes, both eukaryotes and prokaryotes express specialized proteins, transcription factors (TFs). During gene activation, TFs bind gene promotor sequences to recruit the transcriptional machinery including DNA polymerase II. TFs are often multi-subunit proteins containing a DNA-binding domain (DBD) as well as a protein-protein interaction interface. It was suggested that the duration of a TF-DNA binding event 1) depends on these two subunits and 2) dictates the outcome, i.e. the amount of mRNA produced from an activated gene. We set out to address these hypotheses using the transcriptional activator NF-{kappa}B (p65) as well as a number of mutants affecting different functional subunits. Using a combination of live-cell microscopy and RNA sequencing, we show that p65 DNA-binding time indeed correlates with the transcriptional output, but that this relationship depends on, and hence can be uncoupled by altering, the protein-protein interaction capacity. Our results suggest that, while p65 DNA binding times are dominated by the DBD, a transcriptional output can only be achieved with a functional protein-protein interaction subunit.

genetics↗