15 research outputs found
Primordial Black Holes as Generators of Cosmic Structures
Primordial black holes (PBHs) could provide the dark matter in various mass
windows below and those of might explain the
LIGO events. PBHs much larger than this might have important consequences even
if they provide only a small fraction of the dark matter. In particular, they
could generate cosmological structure either individually through through the
`seed' effect or collectively through the `Poisson' effect, thereby alleviating
some problems associated with the standard CDM scenario. If the PBHs all have a
similar mass and make a small contribution to the dark matter, then the seed
effect dominates on small scales, in which case PBHs could seed the
supermassive black holes in galactic nuclei or even galaxies themselves. If
they have a similar mass and provide the dark matter, the Poisson effect
dominates on all scales and the first bound clouds would form earlier than in
the usual scenario, with interesting observational consequences. If the PBHs
have an extended mass spectrum, which is more likely, they could fulfill all
three roles - providing the dark matter, binding the first bound clouds and
generating galaxies. In this case, the galactic mass function naturally has the
observed form, with the galaxy mass being simply related to the black hole
mass. The stochastic gravitational wave background from the PBHs in this
scenario would extend continuously from the LIGO frequency to the LISA
frequency, offering a potential goal for future surveys.Comment: 48 pages, 3 figures, accepted for publication in Monthly Notices of
Royal Astronomical Societ
Phosphoproteomic analysis identifies supervillin as an ERK3 substrate regulating cytokinesis and cell ploidy
Extracellular signal-regulated kinase 3 (ERK3) is a poorly characterized member of the mitogen-activated protein (MAP) kinase family. Functional analysis of the ERK3 signaling pathway has been hampered by a lack of knowledge about the substrates and downstream effectors of the kinase. Here, we used large-scale quantitative phosphoproteomics and targeted gene silencing to identify direct ERK3 substrates and gain insight into its cellular functions. Detailed validation of one candidate substrate identified the gelsolin/villin family member supervillin (SVIL) as a bona fide ERK3 substrate. We show that ERK3 phosphorylates SVIL on Ser245 to regulate myosin II activation and cytokinesis completion in dividing cells. Depletion of SVIL or ERK3 leads to increased cytokinesis failure and multinucleation, a phenotype rescued by wild type SVIL but not by the non-phosphorylatable S245A mutant. Our results unveil a new function of the atypical MAP kinase ERK3 in cell division and the regulation of cell ploidy
Sample Collection Method Bias Effects in Quantitative Phosphoproteomics
Current advances
in selective enrichment, fractionation, and MS
detection of phosphorylated peptides allowed identification and quantitation
of tens of thousands phosphosites from minute amounts of biological
material. One of the major challenges in the field is preserving the
in vivo phosphorylation state of the proteins throughout the sample
preparation workflow. This is typically achieved by using phosphatase
inhibitors and denaturing conditions during cell lysis. Here we determine
if the upstream cell collection techniques could introduce changes
in protein phosphorylation. To evaluate the effect of sample collection
protocols on the global phosphorylation status of the cell, we compared
different sample workflows by metabolic labeling and quantitative
mass spectrometry on <i>Saccharomyces cerevisiae</i> cell
cultures. We identified highly similar phosphopeptides for cells harvested
in ice cold isotonic phosphate buffer, cold ethanol, trichloroacetic
acid, and liquid nitrogen. However, quantitative analyses revealed
that the commonly used phosphate buffer unexpectedly activated signaling
events. Such effects may introduce systematic bias in phosphoproteomics
measurements and biochemical analysis
Sample Collection Method Bias Effects in Quantitative Phosphoproteomics
Current advances
in selective enrichment, fractionation, and MS
detection of phosphorylated peptides allowed identification and quantitation
of tens of thousands phosphosites from minute amounts of biological
material. One of the major challenges in the field is preserving the
in vivo phosphorylation state of the proteins throughout the sample
preparation workflow. This is typically achieved by using phosphatase
inhibitors and denaturing conditions during cell lysis. Here we determine
if the upstream cell collection techniques could introduce changes
in protein phosphorylation. To evaluate the effect of sample collection
protocols on the global phosphorylation status of the cell, we compared
different sample workflows by metabolic labeling and quantitative
mass spectrometry on <i>Saccharomyces cerevisiae</i> cell
cultures. We identified highly similar phosphopeptides for cells harvested
in ice cold isotonic phosphate buffer, cold ethanol, trichloroacetic
acid, and liquid nitrogen. However, quantitative analyses revealed
that the commonly used phosphate buffer unexpectedly activated signaling
events. Such effects may introduce systematic bias in phosphoproteomics
measurements and biochemical analysis
Displacement of N/Q-rich Peptides on TiO<sub>2</sub> Beads Enhances the Depth and Coverage of Yeast Phosphoproteome Analyses
Phosphorylation
is a reversible protein modification that regulates
major cellular processes such as cell division, growth, and differentiation
through highly dynamic and complex signaling pathways. Large-scale
phosphoproteomics analyses have been greatly facilitated using affinity
chromatography such as metal oxide affinity chromatography (e.g.,
TiO<sub>2</sub>), which in combination with mass spectrometry has
enabled unbiased detection and quantification of thousands of phosphorylation
sites in a single experiment. However, global phosphoproteome analyses
do not provide comparable enrichment yields for different model organisms.
While the proportion of phosphopeptides exceed 90% in mammalian cells
using TiO<sub>2</sub>, similar levels have been notoriously difficult
to achieve for yeast or dictylostelium cells. In a systematic study
of TiO<sub>2</sub> using cell extracts from different organisms, we
determined that phosphopeptides are coenriched with peptides containing
repetitive stretches of glutamine and asparagine residues. The proportion
of these nonspecific binders can reach up to 50% in cell extracts
from budding yeast and thus limit the depth and comprehensiveness
of phosphoproteomics analyses. To address this limitation, we developed
an effective method that used decoy amino acids to reduce the extent
of nonspecific peptide binding and improve the recovery and detection
of low abundance phosphopeptides that remained undetected by conventional
TiO<sub>2</sub> enrichment protocols
Displacement of N/Q-rich Peptides on TiO<sub>2</sub> Beads Enhances the Depth and Coverage of Yeast Phosphoproteome Analyses
Phosphorylation
is a reversible protein modification that regulates
major cellular processes such as cell division, growth, and differentiation
through highly dynamic and complex signaling pathways. Large-scale
phosphoproteomics analyses have been greatly facilitated using affinity
chromatography such as metal oxide affinity chromatography (e.g.,
TiO<sub>2</sub>), which in combination with mass spectrometry has
enabled unbiased detection and quantification of thousands of phosphorylation
sites in a single experiment. However, global phosphoproteome analyses
do not provide comparable enrichment yields for different model organisms.
While the proportion of phosphopeptides exceed 90% in mammalian cells
using TiO<sub>2</sub>, similar levels have been notoriously difficult
to achieve for yeast or dictylostelium cells. In a systematic study
of TiO<sub>2</sub> using cell extracts from different organisms, we
determined that phosphopeptides are coenriched with peptides containing
repetitive stretches of glutamine and asparagine residues. The proportion
of these nonspecific binders can reach up to 50% in cell extracts
from budding yeast and thus limit the depth and comprehensiveness
of phosphoproteomics analyses. To address this limitation, we developed
an effective method that used decoy amino acids to reduce the extent
of nonspecific peptide binding and improve the recovery and detection
of low abundance phosphopeptides that remained undetected by conventional
TiO<sub>2</sub> enrichment protocols
Displacement of N/Q-rich Peptides on TiO<sub>2</sub> Beads Enhances the Depth and Coverage of Yeast Phosphoproteome Analyses
Phosphorylation
is a reversible protein modification that regulates
major cellular processes such as cell division, growth, and differentiation
through highly dynamic and complex signaling pathways. Large-scale
phosphoproteomics analyses have been greatly facilitated using affinity
chromatography such as metal oxide affinity chromatography (e.g.,
TiO<sub>2</sub>), which in combination with mass spectrometry has
enabled unbiased detection and quantification of thousands of phosphorylation
sites in a single experiment. However, global phosphoproteome analyses
do not provide comparable enrichment yields for different model organisms.
While the proportion of phosphopeptides exceed 90% in mammalian cells
using TiO<sub>2</sub>, similar levels have been notoriously difficult
to achieve for yeast or dictylostelium cells. In a systematic study
of TiO<sub>2</sub> using cell extracts from different organisms, we
determined that phosphopeptides are coenriched with peptides containing
repetitive stretches of glutamine and asparagine residues. The proportion
of these nonspecific binders can reach up to 50% in cell extracts
from budding yeast and thus limit the depth and comprehensiveness
of phosphoproteomics analyses. To address this limitation, we developed
an effective method that used decoy amino acids to reduce the extent
of nonspecific peptide binding and improve the recovery and detection
of low abundance phosphopeptides that remained undetected by conventional
TiO<sub>2</sub> enrichment protocols
A Cell-Signaling Network Temporally Resolves Specific versus Promiscuous Phosphorylation
If specific and functional kinase- or phosphatase-substrate interactions are optimized for binding compared to promiscuous interactions, then changes in phosphorylation should occur faster on functional versus promiscuous substrates. To test this hypothesis, we designed a high temporal resolution global phosphoproteomics protocol to study the high-osmolarity glycerol (HOG) response in the budding yeast Saccharomyces cerevisiae. The method provides accurate, stimulus-specific measurement of phosphoproteome changes, quantitative analysis of phosphodynamics at sub-minute temporal resolution, and detection of more phosphosites. Rates of evolution of dynamic phosphosites were comparable to those of known functional phosphosites and significantly lower than static or longer-time-frame dynamic phosphosites. Kinetic profile analyses indicated that putatively functional kinase- or phosphatase-substrate interactions occur more rapidly, within 60 s, than promiscuous interactions. Finally, we report many changes in phosphorylation of proteins implicated in cytoskeletal and mitotic spindle dynamics that may underlie regulation of cell cycle and morphogenesis
Combined Enrichment/Enzymatic Approach To Study Tightly Clustered Multisite Phosphorylation on Ser-Rich Domains
The
regulation of protein function through phosphorylation is often
dominated by allosteric interactions and conformational changes. However,
alternative mechanisms involving electrostatic interactions also regulate
protein function. In particular, phosphorylation of clusters of Ser/Thr
residues can affect protein-plasma membrane/chromatin interactions
by electrostatic interactions between phosphosites and phospholipids
or histones. Currently, only a few examples of such mechanisms are
reported, primarily because of the difficulties of detecting highly
phosphorylated proteins and peptides, due in part to the low ionization
efficiency and fragmentation yield of multiphosphorylated peptides
in mass spectrometry when using positive ion mode detection. This
difficulty in detection has resulted in under-reporting of such modified
regions, which can be thought of as phosphoproteomic dark matter.
Here, we present a novel approach that enriches for multisite-phosphorylated
peptides that until now remained inaccessible by conventional phosphoproteomics.
Our technique enables the identification of multisite-phosphorylated
regions on more than 300 proteins in both yeast and human cells and
can be used to profile changes in multisite phosphorylation upon cell
stimulation. We further characterize the role of multisite phosphorylation
for Ste20 in the yeast mating pheromone response. Mutagenesis experiments
confirmed that multisite phosphorylation of Ser/Thr-rich regions plays
an important role in the regulation of Ste20 activity during mating
pheromone signaling. The ability to detect protein multisite phosphorylation
opens new avenues to explore phosphoproteomic dark matter and to study
Ser-rich proteins that interact with binding partners through charge
pairing mechanisms
Combined Enrichment/Enzymatic Approach To Study Tightly Clustered Multisite Phosphorylation on Ser-Rich Domains
The
regulation of protein function through phosphorylation is often
dominated by allosteric interactions and conformational changes. However,
alternative mechanisms involving electrostatic interactions also regulate
protein function. In particular, phosphorylation of clusters of Ser/Thr
residues can affect protein-plasma membrane/chromatin interactions
by electrostatic interactions between phosphosites and phospholipids
or histones. Currently, only a few examples of such mechanisms are
reported, primarily because of the difficulties of detecting highly
phosphorylated proteins and peptides, due in part to the low ionization
efficiency and fragmentation yield of multiphosphorylated peptides
in mass spectrometry when using positive ion mode detection. This
difficulty in detection has resulted in under-reporting of such modified
regions, which can be thought of as phosphoproteomic dark matter.
Here, we present a novel approach that enriches for multisite-phosphorylated
peptides that until now remained inaccessible by conventional phosphoproteomics.
Our technique enables the identification of multisite-phosphorylated
regions on more than 300 proteins in both yeast and human cells and
can be used to profile changes in multisite phosphorylation upon cell
stimulation. We further characterize the role of multisite phosphorylation
for Ste20 in the yeast mating pheromone response. Mutagenesis experiments
confirmed that multisite phosphorylation of Ser/Thr-rich regions plays
an important role in the regulation of Ste20 activity during mating
pheromone signaling. The ability to detect protein multisite phosphorylation
opens new avenues to explore phosphoproteomic dark matter and to study
Ser-rich proteins that interact with binding partners through charge
pairing mechanisms