Pumping up the volume Galbraith, Catherine G
The Journal of cell biology,
02/2023, Letnik:
222, Številka:
2
Journal Article
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The time and cost of annotating ground-truth images and network training are major challenges to utilizing machine learning to automate the mining of volume electron microscopy data. In this issue, ...Gallusser et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202208005) present a less computationally intense pipeline to detect a single type of organelle using a limited number of loosely annotated images.
We demonstrate live-cell super-resolution imaging using photoactivated localization microscopy (PALM). The use of photon-tolerant cell lines in combination with the high resolution and molecular ...sensitivity of PALM permitted us to investigate the nanoscale dynamics within individual adhesion complexes (ACs) in living cells under physiological conditions for as long as 25 min, with half of the time spent collecting the PALM images at spatial resolutions down to approximately 60 nm and frame rates as short as 25 s. We visualized the formation of ACs and measured the fractional gain and loss of individual paxillin molecules as each AC evolved. By allowing observation of a wide variety of nanoscale dynamics, live-cell PALM provides insights into molecular assembly during the initiation, maturation and dissolution of cellular processes.
A key challenge when imaging living cells is how to noninvasively extract the most spatiotemporal information possible. Unlike popular wide-field and confocal methods, plane-illumination microscopy ...limits excitation to the information-rich vicinity of the focal plane, providing effective optical sectioning and high speed while minimizing out-of-focus background and premature photobleaching. Here we used scanned Bessel beams in conjunction with structured illumination and/or two-photon excitation to create thinner light sheets (<0.5 μm) better suited to three-dimensional (3D) subcellular imaging. As demonstrated by imaging the dynamics of mitochondria, filopodia, membrane ruffles, intracellular vesicles and mitotic chromosomes in live cells, the microscope currently offers 3D isotropic resolution down to ∼0.3 μm, speeds up to nearly 200 image planes per second and the ability to noninvasively acquire hundreds of 3D data volumes from single living cells encompassing tens of thousands of image frames.
Super-resolution microscopy at a glance Galbraith, Catherine G; Galbraith, James A
Journal of cell science,
05/2011, Letnik:
124, Številka:
Pt 10
Journal Article
Despite more than 100 years of study, it is unclear if the movement of proteins inside the cell is best described as a mosh pit or an exquisitely choreographed dance. Recent studies suggest the ...latter. Local interactions induce molecular condensates such as liquid-liquid phase separations (LLPSs) or non-liquid, functionally significant molecular aggregates, including synaptic densities, nucleoli, and Amyloid fibrils. Molecular condensates trigger intracellular signaling and drive processes ranging from gene expression to cell division. However, the descriptions of condensates tend to be qualitative and correlative. Here, we indicate how single-molecule imaging and analyses can be applied to quantify condensates. We discuss the pros and cons of different techniques for measuring differences between transient molecular behaviors inside and outside condensates. Finally, we offer suggestions for how imaging and analyses from different time and space regimes can be combined to identify molecular behaviors indicative of condensates within the dynamic high-density intracellular environment.
Migrating cells extend protrusions, probing the surrounding matrix in search of permissive sites to form adhesions. We found that actin fibers polymerizing along the leading edge directed local ...protrusions and drove synchronous sideways movement of β₁ integrin adhesion receptors. These movements lead to the clustering and positioning of conformationally activated, but unligated, β₁ integrins along the leading edge of fibroblast lamellae and growth cone filopodia. Thus, rapid actin-based movement of primed integrins along the leading edge suggests a "sticky fingers" mechanism to probe for new adhesion sites and to direct migration.
Accurate determination of the relative positions of proteins within localized regions of the cell is essential for understanding their biological function. Although fluorescent fusion proteins are ...targeted with molecular precision, the position of these genetically expressed reporters is usually known only to the resolution of conventional optics (almost equal to200 nm). Here, we report the use of two-color photoactivated localization microscopy (PALM) to determine the ultrastructural relationship between different proteins fused to spectrally distinct photoactivatable fluorescent proteins (PA-FPs). The nonperturbative incorporation of these endogenous tags facilitates an imaging resolution in whole, fixed cells of almost equal to20-30 nm at acquisition times of 5-30 min. We apply the technique to image different pairs of proteins assembled in adhesion complexes, the central attachment points between the cytoskeleton and the substrate in migrating cells. For several pairs, we find that proteins that seem colocalized when viewed by conventional optics are resolved as distinct interlocking nano-aggregates when imaged via PALM. The simplicity, minimal invasiveness, resolution, and speed of the technique all suggest its potential to directly visualize molecular interactions within cellular structures at the nanometer scale.
To adhere and migrate, cells must be capable of applying cytoskeletal force to the extracellular matrix (ECM) through integrin receptors. However, it is unclear if connections between integrins and ...the ECM are immediately capable of transducing cytoskeletal contraction into migration force, or whether engagement of force transmission requires maturation of the adhesion. Here, we show that initial integrin-ECM adhesions become capable of exerting migration force with the recruitment of vinculin, a marker for focal complexes, which are precursors of focal adhesions. We are able to induce the development of focal complexes by the application of mechanical force to fibronectin receptors from inside or outside the cell, and we are able to extend focal complex formation to vitronectin receptors by the removal of c-Src. These results indicate that cells use mechanical force as a signal to strengthen initial integrin-ECM adhesions into focal complexes and regulate the amount of migration force applied to individual adhesions at localized regions of the advancing lamella.
Recent advances in light microscopy permit visualization of the behavior of individual molecules within dense macromolecular ensembles in live cells. It is now conceptually possible to relate the ...dynamic organization of molecular machinery to cellular function. However, inherent heterogeneities, as well as disparities between spatial and temporal scales, pose substantial challenges in deriving such a relationship. New approaches are required to link discrete single-molecule behavior with continuous cellular-level processes. Here we combined intercalated molecular and cellular imaging with a computational framework to detect reproducible transient changes in the behavior of individual molecules that are linked to cellular behaviors. Applying our approach to integrin transmembrane receptors revealed a spatial density gradient underlying characteristic molecular density increases and mobility decreases, indicating the subsequent onset of local protrusive activity. Integrin mutants further revealed that these density and mobility transients are separable and depend on different binding domains within the integrin cytoplasmic tail. Our approach provides a generalizable paradigm for dissecting dynamic spatiotemporal molecular behaviors linked to local cellular events.
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