Previous work has established that activation of Mos, Mek, and p42 mitogen-activated protein (MAP... more Previous work has established that activation of Mos, Mek, and p42 mitogen-activated protein (MAP) kinase can trigger release from G 2-phase arrest in Xenopus oocytes and oocyte extracts and can cause Xenopus embryos and extracts to arrest in mitosis. Herein we have found that activation of the MAP kinase cascade can also bring about an interphase arrest in cycling extracts. Activation of the cascade early in the cycle was found to bring about the interphase arrest, which was characterized by an intact nuclear envelope, partially condensed chromatin, and interphase levels of H1 kinase activity, whereas activation of the cascade just before mitosis brought about the mitotic arrest, with a dissolved nuclear envelope, condensed chromatin, and high levels of H1 kinase activity. Early MAP kinase activation did not interfere significantly with DNA replication, cyclin synthesis, or association of cyclins with Cdc2, but it did prevent hyperphosphorylation of Cdc25 and Wee1 and activation of Cdc2/cyclin complexes. Thus, the extracts were arrested in a G 2-like state, unable to activate Cdc2/cyclin complexes. The MAP kinase-induced G 2 arrest appeared not to be related to the DNA replication checkpoint and not to be mediated through inhibition of Cdk2/cyclin E; evidently a novel mechanism underlies this arrest. Finally, we found that by delaying the inactivation of MAP kinase during release of a cytostatic factor-arrested extract from its arrest state, we could delay the subsequent entry into mitosis. This finding suggests that it is the persistence of activated MAP kinase after fertilization that allows the occurrence of a G 2-phase during the first mitotic cell cycle.
Translocation of cytoplasmic molecules to the plasma membrane is commonplace in cell signaling. M... more Translocation of cytoplasmic molecules to the plasma membrane is commonplace in cell signaling. Membrane localization has been hypothesized to increase intermolecular association rates; however, it has also been argued that association should be faster in the cytosol because membrane diffusion is slow. Here we directly compare an identical association reaction in solution and on supported membranes. The measured rate constants show that for 10 μm-radius spherical cell, association is 15-25-fold faster at the membrane than in the cytoplasm. The advantage is cell size-dependent, and for typical ~1 μm prokaryotic cells it should be essentially negligible. Rate enhancement is attributable to a combination of closer proximity of the signaling molecule to its targets after translocation and the higher efficiency of a two-dimensional search.
Ferrell’s group has been exploring the question of how regulatory signals spread through cells. B... more Ferrell’s group has been exploring the question of how regulatory signals spread through cells. By using as model system Xenopus egg extracts, the lab demonstrated that Cdk1 activity which makes mitosis happen and caspase-3/7 which makes apoptosis happen spread through the cytoplasm via what are termed trigger waves. There is good evidence that they do also in intact Xenopus eggs. Trigger waves require only three basic ingredients (positive feedback in the biochemical reactions, a mechanism for local spatial coupling, and a localized initiation point). The Ferrell lab suspects that they will prove to be widespread in the coordination of signaling in large cells and tissues. March 26, 2019 EPFL SV.1717 Trigger waves in cell signaling JaMEs E. FERRELL
Publisher Summary Celenza and Carlson have described a method for assessing the activities of pro... more Publisher Summary Celenza and Carlson have described a method for assessing the activities of protein kinases bound to nitrocellulose-blotting membranes. The method exploits the ability of sodium dodecyl sulfate (SDS)-denatured enzymes to regain activity after treatment with guanidine and nonionic detergent. The resulting kinase activities might arise from disinhibition of partially denatured enzymes or from renaturation of denatured enzymes. This chapter presents a modification of the Celenza and Carlson assay, in which, poly(vinylidene difluoride) (PVDF) membranes are used in place of nitrocellulose. The main advantage of PVDF in this method is that it can be washed with strong bases, which significantly lowers the background of unincorporated radiolabel without diminishing the kinase signal. In addition, milk is not used in the protocol. At least some batches of nonfat dry milk possess detectable levels of kinase activity, which means that blotted proteins that appear to possess kinase activity could actually represent preferred substrates for the milk kinase.
Recent studies by Park, Zarrinipar, and Lim with reengineered Ste5 scaffold proteins underscore t... more Recent studies by Park, Zarrinipar, and Lim with reengineered Ste5 scaffold proteins underscore the fundamental importance of proximity in enzyme regulation and of keeping a proper distance for maintaining signaling specificity.
The fast-growing nontuberculous mycobacterial species Mycobacterium chelonae was isolated from si... more The fast-growing nontuberculous mycobacterial species Mycobacterium chelonae was isolated from six captive South African clawed frogs (Xenopus laevis) with chronic weight loss and nonhealing ulcerative skin lesions. Three of the M. chelonae isolates were evaluated to confirm the species identification using polymerase chain reaction restriction analysis. Disease associated with M. chelonae is reported mainly in people and in fish. To our knowledge, this is the first report of disease associated with M. chelonae in a colony of captive Xenopus sp.
Journal of the American Veterinary Medical Association, Jan 15, 1999
During the summer of 1996, an outbreak of Flavobacterium meningosepticum infection developed in a... more During the summer of 1996, an outbreak of Flavobacterium meningosepticum infection developed in a colony of South African clawed frogs (Xenopus laevis). Clinical signs were consistent with septicemia: ascites, anasarca, dyspnea, extreme lethargy, congestion of web vessels, petechial hemorrhages, and sudden death. Mortality rate reached 35%, and all infections were fatal. The organism was resistant to most antibiotics but was susceptible to enrofloxacin, chloramphenicol, and trimethoprim-sulfadiazine. Treatment with trimethoprim-sulfadiazine was unsuccessful. Although the point source of the infection was not determined, several environmental reservoirs were identified, including a communal water barrel and various pieces of equipment. Molecular strain typing by pulsed-field gel electrophoresis and biochemical analyses revealed that frogs were infected with a single strain of F meningosepticum. Sanitation and management procedures were effective in controlling the outbreak.
The p42 mitogen-activated protein kinase (MAPK) is required for progression through meiotic M pha... more The p42 mitogen-activated protein kinase (MAPK) is required for progression through meiotic M phase in Xenopus oocytes. This report examines whether it also plays a role in normal mitotic progression. MAPK was transiently activated during mitosis in cycling Xenopus egg extracts after activation of the cyclin-dependent kinase Cdc2–cyclin B. Interference with MAPK activation by immunodepletion of its activator MEK, or by addition of the MEK inhibitor PD98059, caused precocious termination of mitosis and interfered with production of normal mitotic microtubules. Sustained activation of MAPK arrested extracts in mitosis in the absence of active Cdc2–cyclin B. These findings identify a role for MEK and MAPK in maintaining the mitotic state.
Recent work has shown that the efficacy of a small interfering RNA (siRNA) for silencing gene exp... more Recent work has shown that the efficacy of a small interfering RNA (siRNA) for silencing gene expression is a function of how easy it is to unwind the siRNA from the 5'-antisense end. Based on these insights, one group has designed an algorithm that substantially improves the odds of picking an effective siRNA, and two groups have shown that 'forked' or 'frayed' siRNAs, which should be easier to unwind from the 5'-antisense end, are more effective than conventional siRNAs. These strategies represent important steps towards the rational design of effective siRNAs.
Persistent activation of p42 mitogen-activated protein kinase (p42 MAPK) during mitosis induces a... more Persistent activation of p42 mitogen-activated protein kinase (p42 MAPK) during mitosis induces a “cytostatic factor” arrest, the arrest responsible for preventing the parthenogenetic activation of unfertilized eggs. The protein kinase p90 Rsk is a substrate of p42 MAPK; thus, the role of p90 Rsk in p42 MAPK-induced mitotic arrest was examined. Xenopus laevis egg extracts immunodepleted of Rsk lost their capacity to undergo mitotic arrest in response to activation of the Mos–MEK-1–p42 MAPK cascade of protein kinases. Replenishing Rsk-depleted extracts with catalytically competent Rsk protein restored the ability of the extracts to undergo mitotic arrest. Rsk appears to be essential for cytostatic factor arrest.
In the context of the bay region, K-region and radical cation hypotheses for polycyclic aromatic ... more In the context of the bay region, K-region and radical cation hypotheses for polycyclic aromatic carcinogens, molecular properties were calculated for fourteen methyl derivatives of benz[a] anthracene (BA) related to (1) intrinsic substrate reactivities towards activating and detoxifying metabolism and (2)-the stabilities of the putative carbocation ultimate carcinogens. Allvalence electron methods were used, avoiding the inherent difficulties found in the ~-electron methods. The calculated substrate reactivities were found to predict major metabolites sucessfuUy, supporting the validity of their use in attempted correlations with observed carcinogenic potencies. Positive correlations were found between observed carcinogenic potencies and (1) the reactivities of the parent polycyclic aromatic hydrocarbons (PAH) towards the initial distal bay region epoxidation and (2) the stabilities of the diol epoxide carbocations. This latter correlation holds when both the methyl derivatives of BA and previously studied unsubstituted PAH are considered together, indicating its potential usefulness as a screening parameter for carcinogenic activity.
When human erythrocytes are incubated with certain phospholipids, the cells become spiculate echi... more When human erythrocytes are incubated with certain phospholipids, the cells become spiculate echinocytes, resembling red cells subjected to metabolic starvation or Ca2+ loading. The present study examines (1) the mode of binding of saturated phosphatidylcholines and egg lysophosphatidylcholine to erythrocytes and (2) the quantitative relationship between phospholipid incorporation and rc;d cell shape. We find that the phospholipids studied become intercalated into erythrocyte membranes, not simply adsorb4 to the cell surface. Spin-labeling and radiolabeling data show that the incorporation of (4 f 1) X lo6 molecules of exogenous phosphatidylcholine per cell converts discocytes to stage 3 echinocytes with about 35 conical spicules. This amount of lipid incorporation is estimated to expand the red cell membrane outer monolayer by 1.7% f 0.6%. Calculations of the inner and outer monolayer surface areas of model discocytes and stage 3 echinocytes yield an estimated difference of 0.7% & 0.2%.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1979
Adrenergic modification of membrane protein phosphorylation was studied in intact human erythrocy... more Adrenergic modification of membrane protein phosphorylation was studied in intact human erythrocytes. Micromolar norepinephrine increased 32p incorporation into Band 2 by 70%, and into Band 3 by 40%. Phosphorylation levels observed with a series of specific agonists and antagonists suggest that an a-adrenergic receptor is involved in this effect. The mechanism of linkage between this receptor and protein phosphorylation does not appear to involve modulation of intracellular concentrations of ATP, cyclic AMP, or cyclic GMP.
Quantitative biochemical studies have shown that the Cdk1/APC system, which drives entry into and... more Quantitative biochemical studies have shown that the Cdk1/APC system, which drives entry into and exit from mitosis, functions as a relaxation oscillator. The bistable switch for the oscillator is provided by the Cdk1/Wee1/Cdc25 subsystem , which consists of a pair of mirror-image positive feedback and double-negative feedback loops. In turn, the bistable switch relies on the ultrasensitive sigmoidal response functions of the two loops' components. Here we have investigated the mechanisms through which Wee1 and Cdc25 generate ultrasensitive responses. Our results argue that the ultrasensitivity arises mainly through cooperative multisite phosphorylation and competition.
A simple, rapid, two-dimensional TLC system is presented which resolves the four phosphoinositide... more A simple, rapid, two-dimensional TLC system is presented which resolves the four phosphoinositide cycle phospholipids as well as all commonly encountered major and minor phospholipids. Ca2+-free lipid samples are loaded onto silica gel HL plates and developed first in 48:40:7:5 chloroform:methanol:water:concentrated ammonia, and then in 55:25:5 chloroform:methanol:formic acid. The method was applied successfully to human erythrocytes, human platelets, and BL/VL3 murine lymphoma cells.
ABSTRACTMouse lemurs are the smallest, fastest reproducing, and among the most abundant primates,... more ABSTRACTMouse lemurs are the smallest, fastest reproducing, and among the most abundant primates, and an emerging model organism for primate biology, behavior, health and conservation. Although much has been learned about their physiology and their Madagascar ecology and phylogeny, little is known about their cellular and molecular biology. Here we used droplet- and plate-based single cell RNA-sequencing to profile 226,000 cells from 27 mouse lemur organs and tissues opportunistically procured from four donors clinically and histologically characterized. Using computational cell clustering, integration, and expert cell annotation, we defined and biologically organized over 750 mouse lemur molecular cell types and their full gene expression profiles. These include cognates of most classical human cell types, including stem and progenitor cells, and the developmental programs for spermatogenesis, hematopoiesis, and other adult tissues. We also described dozens of previously unidentifi...
Mitosis is a dramatic process that affects all parts of the cell. It is driven by an oscillator w... more Mitosis is a dramatic process that affects all parts of the cell. It is driven by an oscillator whose various components are localized in the nucleus, centrosome, and cytoplasm. In principle, the cellular location with the fastest intrinsic rhythm should act as a pacemaker for the process. Here we traced the waves of tubulin polymerization and depolymerization that occur at mitotic entry and exit in Xenopus egg extracts back to their origins. We found that mitosis was commonly initiated at sperm-derived nuclei and their accompanying centrosomes. The cell cycle was~20% faster at these initiation points than in the slowest regions of the extract. Nuclei produced from phage DNA, which did not possess centrosomes, also acted as trigger wave sources, but purified centrosomes in the absence of nuclei did not. We conclude that the nucleus accelerates mitotic entry and propose that it acts as a pacemaker for cell cycle.
Efficient chemotaxis requires rapid coordination between different parts of the cell in response ... more Efficient chemotaxis requires rapid coordination between different parts of the cell in response to changing directional cues. Here we investigate the mechanism of front-rear coordination in chemotactic neutrophils. We find that changes in the protrusion rate at the cell front are instantaneously coupled to changes in retraction at the cell rear, while myosin II accumulation at
Previous work has established that activation of Mos, Mek, and p42 mitogen-activated protein (MAP... more Previous work has established that activation of Mos, Mek, and p42 mitogen-activated protein (MAP) kinase can trigger release from G 2-phase arrest in Xenopus oocytes and oocyte extracts and can cause Xenopus embryos and extracts to arrest in mitosis. Herein we have found that activation of the MAP kinase cascade can also bring about an interphase arrest in cycling extracts. Activation of the cascade early in the cycle was found to bring about the interphase arrest, which was characterized by an intact nuclear envelope, partially condensed chromatin, and interphase levels of H1 kinase activity, whereas activation of the cascade just before mitosis brought about the mitotic arrest, with a dissolved nuclear envelope, condensed chromatin, and high levels of H1 kinase activity. Early MAP kinase activation did not interfere significantly with DNA replication, cyclin synthesis, or association of cyclins with Cdc2, but it did prevent hyperphosphorylation of Cdc25 and Wee1 and activation of Cdc2/cyclin complexes. Thus, the extracts were arrested in a G 2-like state, unable to activate Cdc2/cyclin complexes. The MAP kinase-induced G 2 arrest appeared not to be related to the DNA replication checkpoint and not to be mediated through inhibition of Cdk2/cyclin E; evidently a novel mechanism underlies this arrest. Finally, we found that by delaying the inactivation of MAP kinase during release of a cytostatic factor-arrested extract from its arrest state, we could delay the subsequent entry into mitosis. This finding suggests that it is the persistence of activated MAP kinase after fertilization that allows the occurrence of a G 2-phase during the first mitotic cell cycle.
Translocation of cytoplasmic molecules to the plasma membrane is commonplace in cell signaling. M... more Translocation of cytoplasmic molecules to the plasma membrane is commonplace in cell signaling. Membrane localization has been hypothesized to increase intermolecular association rates; however, it has also been argued that association should be faster in the cytosol because membrane diffusion is slow. Here we directly compare an identical association reaction in solution and on supported membranes. The measured rate constants show that for 10 μm-radius spherical cell, association is 15-25-fold faster at the membrane than in the cytoplasm. The advantage is cell size-dependent, and for typical ~1 μm prokaryotic cells it should be essentially negligible. Rate enhancement is attributable to a combination of closer proximity of the signaling molecule to its targets after translocation and the higher efficiency of a two-dimensional search.
Ferrell’s group has been exploring the question of how regulatory signals spread through cells. B... more Ferrell’s group has been exploring the question of how regulatory signals spread through cells. By using as model system Xenopus egg extracts, the lab demonstrated that Cdk1 activity which makes mitosis happen and caspase-3/7 which makes apoptosis happen spread through the cytoplasm via what are termed trigger waves. There is good evidence that they do also in intact Xenopus eggs. Trigger waves require only three basic ingredients (positive feedback in the biochemical reactions, a mechanism for local spatial coupling, and a localized initiation point). The Ferrell lab suspects that they will prove to be widespread in the coordination of signaling in large cells and tissues. March 26, 2019 EPFL SV.1717 Trigger waves in cell signaling JaMEs E. FERRELL
Publisher Summary Celenza and Carlson have described a method for assessing the activities of pro... more Publisher Summary Celenza and Carlson have described a method for assessing the activities of protein kinases bound to nitrocellulose-blotting membranes. The method exploits the ability of sodium dodecyl sulfate (SDS)-denatured enzymes to regain activity after treatment with guanidine and nonionic detergent. The resulting kinase activities might arise from disinhibition of partially denatured enzymes or from renaturation of denatured enzymes. This chapter presents a modification of the Celenza and Carlson assay, in which, poly(vinylidene difluoride) (PVDF) membranes are used in place of nitrocellulose. The main advantage of PVDF in this method is that it can be washed with strong bases, which significantly lowers the background of unincorporated radiolabel without diminishing the kinase signal. In addition, milk is not used in the protocol. At least some batches of nonfat dry milk possess detectable levels of kinase activity, which means that blotted proteins that appear to possess kinase activity could actually represent preferred substrates for the milk kinase.
Recent studies by Park, Zarrinipar, and Lim with reengineered Ste5 scaffold proteins underscore t... more Recent studies by Park, Zarrinipar, and Lim with reengineered Ste5 scaffold proteins underscore the fundamental importance of proximity in enzyme regulation and of keeping a proper distance for maintaining signaling specificity.
The fast-growing nontuberculous mycobacterial species Mycobacterium chelonae was isolated from si... more The fast-growing nontuberculous mycobacterial species Mycobacterium chelonae was isolated from six captive South African clawed frogs (Xenopus laevis) with chronic weight loss and nonhealing ulcerative skin lesions. Three of the M. chelonae isolates were evaluated to confirm the species identification using polymerase chain reaction restriction analysis. Disease associated with M. chelonae is reported mainly in people and in fish. To our knowledge, this is the first report of disease associated with M. chelonae in a colony of captive Xenopus sp.
Journal of the American Veterinary Medical Association, Jan 15, 1999
During the summer of 1996, an outbreak of Flavobacterium meningosepticum infection developed in a... more During the summer of 1996, an outbreak of Flavobacterium meningosepticum infection developed in a colony of South African clawed frogs (Xenopus laevis). Clinical signs were consistent with septicemia: ascites, anasarca, dyspnea, extreme lethargy, congestion of web vessels, petechial hemorrhages, and sudden death. Mortality rate reached 35%, and all infections were fatal. The organism was resistant to most antibiotics but was susceptible to enrofloxacin, chloramphenicol, and trimethoprim-sulfadiazine. Treatment with trimethoprim-sulfadiazine was unsuccessful. Although the point source of the infection was not determined, several environmental reservoirs were identified, including a communal water barrel and various pieces of equipment. Molecular strain typing by pulsed-field gel electrophoresis and biochemical analyses revealed that frogs were infected with a single strain of F meningosepticum. Sanitation and management procedures were effective in controlling the outbreak.
The p42 mitogen-activated protein kinase (MAPK) is required for progression through meiotic M pha... more The p42 mitogen-activated protein kinase (MAPK) is required for progression through meiotic M phase in Xenopus oocytes. This report examines whether it also plays a role in normal mitotic progression. MAPK was transiently activated during mitosis in cycling Xenopus egg extracts after activation of the cyclin-dependent kinase Cdc2–cyclin B. Interference with MAPK activation by immunodepletion of its activator MEK, or by addition of the MEK inhibitor PD98059, caused precocious termination of mitosis and interfered with production of normal mitotic microtubules. Sustained activation of MAPK arrested extracts in mitosis in the absence of active Cdc2–cyclin B. These findings identify a role for MEK and MAPK in maintaining the mitotic state.
Recent work has shown that the efficacy of a small interfering RNA (siRNA) for silencing gene exp... more Recent work has shown that the efficacy of a small interfering RNA (siRNA) for silencing gene expression is a function of how easy it is to unwind the siRNA from the 5'-antisense end. Based on these insights, one group has designed an algorithm that substantially improves the odds of picking an effective siRNA, and two groups have shown that 'forked' or 'frayed' siRNAs, which should be easier to unwind from the 5'-antisense end, are more effective than conventional siRNAs. These strategies represent important steps towards the rational design of effective siRNAs.
Persistent activation of p42 mitogen-activated protein kinase (p42 MAPK) during mitosis induces a... more Persistent activation of p42 mitogen-activated protein kinase (p42 MAPK) during mitosis induces a “cytostatic factor” arrest, the arrest responsible for preventing the parthenogenetic activation of unfertilized eggs. The protein kinase p90 Rsk is a substrate of p42 MAPK; thus, the role of p90 Rsk in p42 MAPK-induced mitotic arrest was examined. Xenopus laevis egg extracts immunodepleted of Rsk lost their capacity to undergo mitotic arrest in response to activation of the Mos–MEK-1–p42 MAPK cascade of protein kinases. Replenishing Rsk-depleted extracts with catalytically competent Rsk protein restored the ability of the extracts to undergo mitotic arrest. Rsk appears to be essential for cytostatic factor arrest.
In the context of the bay region, K-region and radical cation hypotheses for polycyclic aromatic ... more In the context of the bay region, K-region and radical cation hypotheses for polycyclic aromatic carcinogens, molecular properties were calculated for fourteen methyl derivatives of benz[a] anthracene (BA) related to (1) intrinsic substrate reactivities towards activating and detoxifying metabolism and (2)-the stabilities of the putative carbocation ultimate carcinogens. Allvalence electron methods were used, avoiding the inherent difficulties found in the ~-electron methods. The calculated substrate reactivities were found to predict major metabolites sucessfuUy, supporting the validity of their use in attempted correlations with observed carcinogenic potencies. Positive correlations were found between observed carcinogenic potencies and (1) the reactivities of the parent polycyclic aromatic hydrocarbons (PAH) towards the initial distal bay region epoxidation and (2) the stabilities of the diol epoxide carbocations. This latter correlation holds when both the methyl derivatives of BA and previously studied unsubstituted PAH are considered together, indicating its potential usefulness as a screening parameter for carcinogenic activity.
When human erythrocytes are incubated with certain phospholipids, the cells become spiculate echi... more When human erythrocytes are incubated with certain phospholipids, the cells become spiculate echinocytes, resembling red cells subjected to metabolic starvation or Ca2+ loading. The present study examines (1) the mode of binding of saturated phosphatidylcholines and egg lysophosphatidylcholine to erythrocytes and (2) the quantitative relationship between phospholipid incorporation and rc;d cell shape. We find that the phospholipids studied become intercalated into erythrocyte membranes, not simply adsorb4 to the cell surface. Spin-labeling and radiolabeling data show that the incorporation of (4 f 1) X lo6 molecules of exogenous phosphatidylcholine per cell converts discocytes to stage 3 echinocytes with about 35 conical spicules. This amount of lipid incorporation is estimated to expand the red cell membrane outer monolayer by 1.7% f 0.6%. Calculations of the inner and outer monolayer surface areas of model discocytes and stage 3 echinocytes yield an estimated difference of 0.7% & 0.2%.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1979
Adrenergic modification of membrane protein phosphorylation was studied in intact human erythrocy... more Adrenergic modification of membrane protein phosphorylation was studied in intact human erythrocytes. Micromolar norepinephrine increased 32p incorporation into Band 2 by 70%, and into Band 3 by 40%. Phosphorylation levels observed with a series of specific agonists and antagonists suggest that an a-adrenergic receptor is involved in this effect. The mechanism of linkage between this receptor and protein phosphorylation does not appear to involve modulation of intracellular concentrations of ATP, cyclic AMP, or cyclic GMP.
Quantitative biochemical studies have shown that the Cdk1/APC system, which drives entry into and... more Quantitative biochemical studies have shown that the Cdk1/APC system, which drives entry into and exit from mitosis, functions as a relaxation oscillator. The bistable switch for the oscillator is provided by the Cdk1/Wee1/Cdc25 subsystem , which consists of a pair of mirror-image positive feedback and double-negative feedback loops. In turn, the bistable switch relies on the ultrasensitive sigmoidal response functions of the two loops' components. Here we have investigated the mechanisms through which Wee1 and Cdc25 generate ultrasensitive responses. Our results argue that the ultrasensitivity arises mainly through cooperative multisite phosphorylation and competition.
A simple, rapid, two-dimensional TLC system is presented which resolves the four phosphoinositide... more A simple, rapid, two-dimensional TLC system is presented which resolves the four phosphoinositide cycle phospholipids as well as all commonly encountered major and minor phospholipids. Ca2+-free lipid samples are loaded onto silica gel HL plates and developed first in 48:40:7:5 chloroform:methanol:water:concentrated ammonia, and then in 55:25:5 chloroform:methanol:formic acid. The method was applied successfully to human erythrocytes, human platelets, and BL/VL3 murine lymphoma cells.
ABSTRACTMouse lemurs are the smallest, fastest reproducing, and among the most abundant primates,... more ABSTRACTMouse lemurs are the smallest, fastest reproducing, and among the most abundant primates, and an emerging model organism for primate biology, behavior, health and conservation. Although much has been learned about their physiology and their Madagascar ecology and phylogeny, little is known about their cellular and molecular biology. Here we used droplet- and plate-based single cell RNA-sequencing to profile 226,000 cells from 27 mouse lemur organs and tissues opportunistically procured from four donors clinically and histologically characterized. Using computational cell clustering, integration, and expert cell annotation, we defined and biologically organized over 750 mouse lemur molecular cell types and their full gene expression profiles. These include cognates of most classical human cell types, including stem and progenitor cells, and the developmental programs for spermatogenesis, hematopoiesis, and other adult tissues. We also described dozens of previously unidentifi...
Mitosis is a dramatic process that affects all parts of the cell. It is driven by an oscillator w... more Mitosis is a dramatic process that affects all parts of the cell. It is driven by an oscillator whose various components are localized in the nucleus, centrosome, and cytoplasm. In principle, the cellular location with the fastest intrinsic rhythm should act as a pacemaker for the process. Here we traced the waves of tubulin polymerization and depolymerization that occur at mitotic entry and exit in Xenopus egg extracts back to their origins. We found that mitosis was commonly initiated at sperm-derived nuclei and their accompanying centrosomes. The cell cycle was~20% faster at these initiation points than in the slowest regions of the extract. Nuclei produced from phage DNA, which did not possess centrosomes, also acted as trigger wave sources, but purified centrosomes in the absence of nuclei did not. We conclude that the nucleus accelerates mitotic entry and propose that it acts as a pacemaker for cell cycle.
Efficient chemotaxis requires rapid coordination between different parts of the cell in response ... more Efficient chemotaxis requires rapid coordination between different parts of the cell in response to changing directional cues. Here we investigate the mechanism of front-rear coordination in chemotactic neutrophils. We find that changes in the protrusion rate at the cell front are instantaneously coupled to changes in retraction at the cell rear, while myosin II accumulation at
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Papers by James Ferrell