4,680 research outputs found
Millisecond single-molecule localization microscopy combined with convolution analysis and automated image segmentation to determine protein concentrations in complexly structured, functional cells, one cell at a time
We present a single-molecule tool called the CoPro (Concentration of
Proteins) method that uses millisecond imaging with convolution analysis,
automated image segmentation and super-resolution localization microscopy to
generate robust estimates for protein concentration in different compartments
of single living cells, validated using realistic simulations of complex
multiple compartment cell types. We demonstrates its utility experimentally on
model Escherichia coli bacteria and Saccharomyces cerevisiae budding yeast
cells, and use it to address the biological question of how signals are
transduced in cells. Cells in all domains of life dynamically sense their
environment through signal transduction mechanisms, many involving gene
regulation. The glucose sensing mechanism of S. cerevisiae is a model system
for studying gene regulatory signal transduction. It uses the multi-copy
expression inhibitor of the GAL gene family, Mig1, to repress unwanted genes in
the presence of elevated extracellular glucose concentrations. We fluorescently
labelled Mig1 molecules with green fluorescent protein (GFP) via chromosomal
integration at physiological expression levels in living S. cerevisiae cells,
in addition to the RNA polymerase protein Nrd1 with the fluorescent protein
reporter mCherry. Using CoPro we make quantitative estimates of Mig1 and Nrd1
protein concentrations in the cytoplasm and nucleus compartments on a
cell-by-cell basis under physiological conditions. These estimates indicate a
4-fold shift towards higher values in concentration of diffusive Mig1 in the
nucleus if the external glucose concentration is raised, whereas equivalent
levels in the cytoplasm shift to smaller values with a relative change an order
of magnitude smaller. This compares with Nrd1 which is not involved directly in
glucose sensing, which is almost exclusively localized in the nucleus under
high and..
Three-dimensional structure and flexibility of a membrane-coating module of the nuclear pore complex.
The nuclear pore complex mediates nucleocytoplasmic transport in all eukaryotes and is among the largest cellular assemblies of proteins, collectively known as nucleoporins. Nucleoporins are organized into distinct subcomplexes. We optimized the isolation of a putative membrane-coating subcomplex of the nuclear pore complex, the heptameric Nup84 complex, and analyzed its structure by EM. Our data confirmed the previously reported 'Y' shape. We discerned additional structural details, including specific hinge regions at which the particle shows great flexibility. We determined the three-dimensional structures of two conformers, mapped the localization of two nucleoporins within the subcomplex and docked known crystal structures into the EM maps. The free ends of the Y-shaped particle are formed by beta-propellers; the connecting segments consist of alpha-solenoids. Notably, the same organizational principle is found in the clathrin triskelion, which may share a common evolutionary origin with the heptameric complex
ESCRT machinery mediates selective microautophagy of endoplasmic reticulum in yeast
ER-phagy, the selective autophagy of endoplasmic reticulum (ER), safeguards organelle homeostasis by eliminating misfolded proteins and regulating ER size. ER-phagy can occur by macroautophagic and microautophagic mechanisms. While dedicated machinery for macro-ER-phagy has been discovered, the molecules and mechanisms mediating micro-ER-phagy remain unknown. Here, we first show that micro-ER-phagy in yeast involves the conversion of stacked cisternal ER into multilamellar ER whorls during microautophagic uptake into lysosomes. Second, we identify the conserved Nem1-Spo7 phosphatase complex and the ESCRT machinery as key components for micro-ER-phagy. Third, we demonstrate that macro- and micro-ER-phagy are parallel pathways with distinct molecular requirements. Finally, we provide evidence that the ESCRT machinery directly functions in scission of the lysosomal membrane to complete the microautophagic uptake of ER. These findings establish a framework for a mechanistic understanding of micro-ER-phagy and, thus, a comprehensive appreciation of the role of autophagy in ER homeostasis
Systematic Definition of Protein Constituents along the Major Polarization Axis Reveals an Adaptive Reuse of the Polarization Machinery in Pheromone-Treated Budding Yeast
Polarizing cells extensively restructure cellular components in a spatially and temporally coupledmanner along the major axis of cellular extension. Budding yeast are a useful model of polarized growth, helping to define many molecular components of this conserved process. Besides budding, yeast cells also differentiate upon treatment with pheromone from the opposite mating type, forming a mating projection (the ‘shmoo’) by directional restructuring of the cytoskeleton, localized vesicular transport and overall reorganization of the cytosol. To characterize the proteomic localization changes ac-companying polarized growth, we developed and implemented a novel cell microarray-based imaging assay for measuring the spatial redistribution of a large fraction of the yeast proteome, and applied this assay to identify proteins localized along the mating projection following pheromone treatment. We further trained a machine learning algorithm to refine the cell imaging screen, identifying additional shmoo-localized proteins. In all, we identified 74 proteins that specifically localize to the mating projection, including previously uncharacterized proteins (Ycr043c, Ydr348c, Yer071c, Ymr295c, and Yor304c-a) and known polarization complexes such as the exocyst. Functional analysis of these proteins, coupled with quantitative analysis of individual organelle movements during shmoo formation, suggests a model in which the basic machinery for cell polarization is generally conserved between processe
Integrating high-throughput genetic interaction mapping and high-content screening to explore yeast spindle morphogenesis
A combination of yeast genetics, synthetic genetic array analysis, and high-throughput screening reveals that sumoylation of Mcm21p promotes disassembly of the mitotic spindle
Localization of protein aggregation in Escherichia coli is governed by diffusion and nucleoid macromolecular crowding effect
Aggregates of misfolded proteins are a hallmark of many age-related diseases.
Recently, they have been linked to aging of Escherichia coli (E. coli) where
protein aggregates accumulate at the old pole region of the aging bacterium.
Because of the potential of E. coli as a model organism, elucidating aging and
protein aggregation in this bacterium may pave the way to significant advances
in our global understanding of aging. A first obstacle along this path is to
decipher the mechanisms by which protein aggregates are targeted to specific
intercellular locations. Here, using an integrated approach based on
individual-based modeling, time-lapse fluorescence microscopy and automated
image analysis, we show that the movement of aging-related protein aggregates
in E. coli is purely diffusive (Brownian). Using single-particle tracking of
protein aggregates in live E. coli cells, we estimated the average size and
diffusion constant of the aggregates. Our results evidence that the aggregates
passively diffuse within the cell, with diffusion constants that depend on
their size in agreement with the Stokes-Einstein law. However, the aggregate
displacements along the cell long axis are confined to a region that roughly
corresponds to the nucleoid-free space in the cell pole, thus confirming the
importance of increased macromolecular crowding in the nucleoids. We thus used
3d individual-based modeling to show that these three ingredients (diffusion,
aggregation and diffusion hindrance in the nucleoids) are sufficient and
necessary to reproduce the available experimental data on aggregate
localization in the cells. Taken together, our results strongly support the
hypothesis that the localization of aging-related protein aggregates in the
poles of E. coli results from the coupling of passive diffusion- aggregation
with spatially non-homogeneous macromolecular crowding. They further support
the importance of "soft" intracellular structuring (based on macromolecular
crowding) in diffusion-based protein localization in E. coli.Comment: PLoS Computational Biology (2013
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