The piwi/argonaute family of proteins is involved in key developmental processes such as stem cel... more The piwi/argonaute family of proteins is involved in key developmental processes such as stem cell maintenance and axis specification through molecular mechanisms that may involve RNA silencing. Here we report on the cloning and characterization of the sea urchin piwi/argonaute family member seawi. Seawi is a major component of microtubule-ribonucleoprotein (MT-RNP) complexes isolated from two different species of sea urchin, Strongylocentrotus purpuratus and Paracentrotus lividus. Seawi co-isolates with purified ribosomes, cosediments with 80S ribosomes in sucrose density gradients, and binds microtubules. Seawi possesses the RNA binding motif common to piwi family members and binds P. lividus bep4 mRNA, a transcript that co-isolates with MT-RNP complexes and whose translation product has been shown to play a role in patterning the animal-vegetal axis. Indirect immunofluorescence studies localized seawi to the cortex of unfertilized eggs within granule-like particles, the mitotic s...
Mutations in the APC or β-catenin genes are well-established initiators of colorectal cancer, yet... more Mutations in the APC or β-catenin genes are well-established initiators of colorectal cancer, yet modifiers that facilitate the survival and progression of nascent tumor cells are not well defined. Using genetic and pharmacologic approaches in mouse colorectal cancer and human colorectal cancer xenograft models, we show that incipient intestinal tumor cells activate CDC42, an APC-interacting small GTPase, as a crucial step in malignant progression. In the mouse, Cdc42 ablation attenuated the tumorigenicity of mutant intestinal cells carrying single APC or β-catenin mutations. Similarly, human colorectal cancer with relatively higher levels of CDC42 activity was particularly sensitive to CDC42 blockade. Mechanistic studies suggested that Cdc42 may be activated at different levels, including at the level of transcriptional activation of the stem cell-enriched Rho family exchange factor Arhgef4. Our results indicate that early-stage mutant intestinal epithelial cells must recruit the p...
One of the most spectacular motions is the generation of the acrosomal process in the Limulus spe... more One of the most spectacular motions is the generation of the acrosomal process in the Limulus sperm . On contact with the egg, the sperm generates a 60-,um-long process that literally drills its way through the jelly surrounding the egg. This irreversible reaction takes only a few seconds. We suggested earlier that this motion is driven by a change in twist of the actin filaments comprising the acrosomal process. In this paper we analyze the so-called false discharge, a reversible reaction, in which the acrosomal filament bundle extends laterally from the base of the sperm and not anteriorly from the apex . Unlike the true discharge, which is straight, the false discharge is helical. Before extension, the filament bundle is coiled about the base of the sperm. In the coil, the bundle is not smoothly bent but consists of arms (straight segments) and elbows (corners) so that the coil looks like a 14-sided polygon . The extension of the false discharge works as follows: Starting at the basal end of the bundle, the filaments change their twist which concomitantly changes the orientations of the elbows relative to each other; that is, in the coil, the elbows all lie in a common plane, but, after the change in twist, the plane of each elbow is rotated to be perpendicular to that of its neighbors . This change transforms the bundle from a compact coil into an extended left-handed helix. Because the basal end of the bundle is unconstrained, the extension is lateral . The true discharge works the same way but starts at the apical end of the bundle . The apical end, however, is constrained by its passage through the nuclear canal, which directs the extension anteriorly . Unlike the false discharge, during the true discharge the elbows are melted out, making the reaction irreversible . This study shows that rapid movement can be generated by actin without myosin and gives us insight into the molecular mechanism.
Fertilization of sea urchin eggs results in the rapid polymerization of actin filaments and subse... more Fertilization of sea urchin eggs results in the rapid polymerization of actin filaments and subsequent formation of a brush border-like cortical cytoskeleton. A 110 × 10(3) Mr (110K) actin binding protein has been purified from extracts of unfertilized Strongylocentrotus purpuratus eggs. Analysis of polymerization kinetics using fluorescence and viscometry assays demonstrated that 110K accelerated the nucleation phase of actin assembly only in the presence of elevated Ca2+. The Ca(2+)-mediated effects were correlated with a decrease in sedimentable polymer and a decrease in average filament length. Addition of Ca2+ to solutions of 110K and F-actin, polymerized in the presence of EGTA, resulted in a precipitous drop in viscosity and the decreased viscosity was fully reversible upon chelation of Ca2+. The Ca2+ threshold for 110K activation was in the 10(−6) to 10(−7) M range. Nucleated assembly experiments using Limulus sperm acrosomal processes demonstrated that egg 110K capped the b...
Individual human sperm can be micromanipulated in three dimensions using a 1.06 microns Nd:YAG la... more Individual human sperm can be micromanipulated in three dimensions using a 1.06 microns Nd:YAG laser trap. Single sperm swimming with velocities in the range of 65 to 85 microns/sec can be trapped with 40 mW of power through 120 seconds without a deleterious effect on velocity. Even though it will be necessary to further evaluate the effects of laser light on specific functions of sperm, our data suggest that decreasing the time of manipulation to a minimum will increase the safety of the micromanipulation procedures. Laser traps may play a role in assisted reproductive technology by facilitating the selective transport of individual sperm.
Sea urchin coelomocytes undergo an inducible structural transformation from petalloid to filopodi... more Sea urchin coelomocytes undergo an inducible structural transformation from petalloid to filopodial form during the 'clotting' response in sea urchins. Using a petalloid coelomocyte model, stimulated coelomocytes exhibited bidirectional particle/vesicle motility with a broad distribution of velocities, ranging from 0.02 to 0.12 microns s-1 in the outward bound direction. Coelomocytes treated with the microtubule-disrupting drug, nocodazole, continued to exhibit outward particle/vesicle movements along linear paths with an average velocity of 0.028 +/- 0.006 microns s-1. We partially purified a 110 kDa polypeptide possessing K+EDTA-, Ca2(+)-, Mg2(+)- and F-actin-activated Mg(2+)-ATPase activities characteristic of myosin-like motor proteins. The 110 kDa protein immuno-crossreacted with both affinity-purified, anti-brush border unconventional myosin-I polyclonal antibodies and anti-Acanthamoeba myosin head monoclonal antibodies. By indirect immunofluorescence, the 110 kDa unco...
The motile behavior of epithelial cells located at the edge of a large wound in a monolayer of cu... more The motile behavior of epithelial cells located at the edge of a large wound in a monolayer of cultured cells was analyzed. The initial cellular response is alignment of the edge with an accompanying formation of tangential marginal actin bundles within individual cells positioned along the wound edge. Later, coherent out-growths of cell masses occur by the formation of special ''leader'' cells at the tops of outgrowths and ''follower'' cells along the sides. Leader cells exhibit profound cytoskeletal reorganization, including disassembly of marginal bundles, the realignment of actin filament bundles, and penetration of microtubules into highly active lamellae. Additionally, cell-cell contacts acquire radial geometry indicative of increased contractile tension. Interestingly, leader cells acquire a cytoskeletal organization and motility typical of fibroblasts. IAR-2 cultures stably transfected with a dominant-negative mutant of RhoA or treated with Rho-kinase inhibitor Y-27632 transformed most edge cells into leader-like cells. Alternatively , transfection of cells with constitutively active RhoA suppressed formation of leaders. Thus, expansion of the epithelial sheet involves functional differentiation into two distinct types of edge cells. The transition between these two patterns is controlled by Rho activity, which in turn controls the dynamic distribution and activity of actin filament bundles, myosin II, and microtubules.
We have investigated the Ca2+ -dependent interactions of villin, a protein of the intestinal micr... more We have investigated the Ca2+ -dependent interactions of villin, a protein of the intestinal microvillar core, with actin by monitoring resonance energy transfer between fluorescently labeled actin subunits. In the presence of elevated free Ca2+ (approximately 20 microM), villin affects both the nucleation and the elongation phases of actin polymerization. Consistent with previous reports, villin stimulates the nucleation process and will form stable nuclei under depolymerization conditions. Compared to the control, the net rate of polymerization is slightly inhibited at low concentrations of villin (villin/actin approximately 1:400) but is stimulated at higher concentrations (villin/actin greater than 1:100). Villin also significantly increases the critical concentration of actin polymerization. Addition of either villin or villin-actin complexes induces depolymerization of preassembled actin filaments. This villin-induced depolymerization is reversible upon removal of free Ca2+ or...
We used Limulus sperm acrosomal actin bundles to examine the effect of 2 uM cytochalasin B (CB) o... more We used Limulus sperm acrosomal actin bundles to examine the effect of 2 uM cytochalasin B (CB) on elongation from both the barbed and pointed ends of the actin filament. In this paper we report that 2 uM CB does not prevent monomer addition onto the barbed ends of the acrosomal actin filaments. Barbed end assembly occurred over a range of actin monomer concentrations (0.2-6 uM) in solutions conraining 75 mM KCI, 5 mM MgC12, 10 mM Imidazole, pH 7.2, and 2 uM CB. However, the elongation rates were reduced such that the rates at the barbed end were approximately the same as those at the pointed end. The association and dissociation rate constants
We have re-examined the Ca++-dependent interaction of an intestinal microvillar 95-kdalton protei... more We have re-examined the Ca++-dependent interaction of an intestinal microvillar 95-kdalton protein (MV-95K) and actin using the isolated acrosomal process bundles from Limulus sperm. Making use of the processes as nuclei for assembling actin filaments, we quantitatively and qualitatively examined MV-95K's effect on filament assembly and on Factin, both in the presence and in the absence of Ca ++. The acrosomal processes are particularly advantageous for this approach because they nucleate large numbers of filaments, they are extremely stable, and their morphology can be used to determine the polarity of any nulceated filaments. When filament nucleation was initiated in the presence of MV-95K and the absence of Ca +÷ , there was biased filament assembly from the bundle ends. The calculated elongation rates from both the barbed and pointed filament ends were virtually indistinguishable from control preparations. In the presence of Ca ÷+, MV-95K completely inhibited filament assembly from the barbed filament end without affecting the initial rate of assembly from the pointed filament end. The inhibition of assembly results from MV-95K binding to and capping the barbed filament end, thereby preventing monomer addition. This indicates that, while MV-95K is a potent nucleator of actin assembly, it is also a potent inhibitor of actin filament elongation To examine the effects of MV-95K on F-actin in the presence of Ca ++, we developed an assay where MV-95k is added to filaments previously assembled from acrosomal processes without causing filament breakage during mixing. These results clearly demonstrated that rapid filament shortening by MV-95K results through a mechanism of disrupting intrafilament monomermonomer interactions. Finally, we show that tropomyosin-containing actin filaments are insensitive to cutting, but not to capping, by MV-95K in the presence of Ca ++.
The piwi/argonaute family of proteins is involved in key developmental processes such as stem cel... more The piwi/argonaute family of proteins is involved in key developmental processes such as stem cell maintenance and axis specification through molecular mechanisms that may involve RNA silencing. Here we report on the cloning and characterization of the sea urchin piwi/argonaute family member seawi. Seawi is a major component of microtubule-ribonucleoprotein (MT-RNP) complexes isolated from two different species of sea urchin, Strongylocentrotus purpuratus and Paracentrotus lividus. Seawi co-isolates with purified ribosomes, cosediments with 80S ribosomes in sucrose density gradients, and binds microtubules. Seawi possesses the RNA binding motif common to piwi family members and binds P. lividus bep4 mRNA, a transcript that co-isolates with MT-RNP complexes and whose translation product has been shown to play a role in patterning the animal-vegetal axis. Indirect immunofluorescence studies localized seawi to the cortex of unfertilized eggs within granule-like particles, the mitotic s...
Mutations in the APC or β-catenin genes are well-established initiators of colorectal cancer, yet... more Mutations in the APC or β-catenin genes are well-established initiators of colorectal cancer, yet modifiers that facilitate the survival and progression of nascent tumor cells are not well defined. Using genetic and pharmacologic approaches in mouse colorectal cancer and human colorectal cancer xenograft models, we show that incipient intestinal tumor cells activate CDC42, an APC-interacting small GTPase, as a crucial step in malignant progression. In the mouse, Cdc42 ablation attenuated the tumorigenicity of mutant intestinal cells carrying single APC or β-catenin mutations. Similarly, human colorectal cancer with relatively higher levels of CDC42 activity was particularly sensitive to CDC42 blockade. Mechanistic studies suggested that Cdc42 may be activated at different levels, including at the level of transcriptional activation of the stem cell-enriched Rho family exchange factor Arhgef4. Our results indicate that early-stage mutant intestinal epithelial cells must recruit the p...
Sea urchin coelomocytes undergo an inducible structural transformation from petalloid to filopodi... more Sea urchin coelomocytes undergo an inducible structural transformation from petalloid to filopodial form during the 'clotting' response in sea urchins. Using a petalloid coelomocyte model, stimulated coelomocytes exhibited bidirectional particle/vesicle motility with a broad distribution of velocities, ranging from 0.02 to 0.12 microns s-1 in the outward bound direction. Coelomocytes treated with the microtubule-disrupting drug, nocodazole, continued to exhibit outward particle/vesicle movements along linear paths with an average velocity of 0.028 +/- 0.006 microns s-1. We partially purified a 110 kDa polypeptide possessing K+EDTA-, Ca2(+)-, Mg2(+)- and F-actin-activated Mg(2+)-ATPase activities characteristic of myosin-like motor proteins. The 110 kDa protein immuno-crossreacted with both affinity-purified, anti-brush border unconventional myosin-I polyclonal antibodies and anti-Acanthamoeba myosin head monoclonal antibodies. By indirect immunofluorescence, the 110 kDa unco...
The effect of villin on the critical concentration of actin and on the kinetics of its polymeriza... more The effect of villin on the critical concentration of actin and on the kinetics of its polymerization has been measured. In the presence of villin and 10 microM calcium, the critical concentration of actin increased from 0.2 to 0.9 microM. This effect of villin on the critical concentration was shown to be the result of its well-documented ability to block the "barbed" end of actin filaments, i.e., the "high-affinity end" of a polymer with a different monomer binding constant at each end. Thus, below 0.8 microM actin polymerization was prevented when the ratio of villin to actin was about 1 in 1000. Furthermore, the effect of villin was saturable; i.e., the critical concentration remained constant with increasing villin concentration once the maximal change had been obtained. In addition, fragmentation of actin filaments previously capped with villin, producing uncapped filaments, caused a rapid, transient fall of the monomer concentration. With the disappearance of the uncapped filaments the actin monomer concentration returned to that measured before fragmentation. The binding constant of villin to the barbed end of the actin filament was calculated to be greater than 10(11) M-1. The rate constants of elongation and of depolymerization at each end of an actin filament were measured. The depolymerization rate constant from the barbed end was about 10 times greater under conditions leading to complete depolymerization than under steady-state conditions. We discuss a possible explanation for the finding and its implication for possible regulatory mechanisms.
"Cutting" of actin filaments by villin was evaluated from the time course of fi... more "Cutting" of actin filaments by villin was evaluated from the time course of filament depolymerization. Depolymerization was initiated by diluting polymerized actin, labeled with a fluorescent probe on either lysine-374 or cysteine-375, to a concentration well below the critical into a medium containing free villin and various concentrations of calcium (in addition to potassium and magnesium). It was observed that at high calcium concentrations (200 microM) the time course of depolymerization could not be described by the single exponential that defines it at low calcium and low villin levels. Instead, at high calcium, the exponent increased with time and the rate of depolymerization became greater than that of controls in the absence of villin. This contrasts with the inhibition of depolymerization by villin at low calcium. The latter inhibition is a consequence of the capping of the barbed filament end by villin as are the inhibition of filament elongation and the elevation of the critical concentration. Evidence is presented that the effects of villin at high calcium are the result of cutting of the actin filaments by villin. It thus appears that different calcium binding sites control capping and cutting and that the calcium binding sites regulating cutting have a much lower affinity for calcium than the sites regulating capping of the barbed filament ends.
Communication between stem and niche supporting cells maintains the homeostasis of adult tissues.... more Communication between stem and niche supporting cells maintains the homeostasis of adult tissues. Wnt signaling is a crucial regulator of the stem cell niche, but the mechanism that governs Wnt ligand delivery in this compartment has not been fully investigated. We identified that Wnt secretion is partly dependent on Rab8a-mediated anterograde transport of Gpr177 (wntless), a Wnt-specific transmembrane transporter. Gpr177 binds to Rab8a, depletion of which compromises Gpr177 traffic, thereby weakening the secretion of multiple Wnts. Analyses of generic Wnt/β-catenin targets in Rab8a knockout mouse intestinal crypts indicate reduced signaling activities; maturation of Paneth cells - a Wnt-dependent cell type - is severely affected. Rab8a knockout crypts show an expansion of Lgr5(+) and Hopx(+) cells in vivo. However, in vitro, the knockout enteroids exhibit significantly weakened growth that can be partly restored by exogenous Wnts or Gsk3β inhibitors. Immunogold labeling and surface...
The actin cytoskeleton is an integral component of the cell-cell adherens junction complex. We us... more The actin cytoskeleton is an integral component of the cell-cell adherens junction complex. We used fluorescence labeling of actin filaments and time-lapse laser scanning confocal microscopy to investigate the functional relationship between the organization of the actin cytoskeleton and formation of adherens junctions in live epithelial cells. Rhodamine-phalloidin was loaded into cultured cells by wounding epithelial monolayers in the presence of fluorescent analog. Rhodamine-phalloidin was incorporated into the actin filaments in stress fibers, circumferential bundles, and marginal bundles. Cells containing labeled actin filaments appeared physiologically normal since the rates of migration, rates of pseudopodial protrusion/retraction, ability to form contacts, and sensitivity to cytochalasin B were equivalent to non-loaded, control epithelial cells. Marginal actin bundles initially formed as bow-shaped bundles that were observed to straighten as the bundles flowed rearward and aw...
The piwi/argonaute family of proteins is involved in key developmental processes such as stem cel... more The piwi/argonaute family of proteins is involved in key developmental processes such as stem cell maintenance and axis specification through molecular mechanisms that may involve RNA silencing. Here we report on the cloning and characterization of the sea urchin piwi/argonaute family member seawi. Seawi is a major component of microtubule-ribonucleoprotein (MT-RNP) complexes isolated from two different species of sea urchin, Strongylocentrotus purpuratus and Paracentrotus lividus. Seawi co-isolates with purified ribosomes, cosediments with 80S ribosomes in sucrose density gradients, and binds microtubules. Seawi possesses the RNA binding motif common to piwi family members and binds P. lividus bep4 mRNA, a transcript that co-isolates with MT-RNP complexes and whose translation product has been shown to play a role in patterning the animal-vegetal axis. Indirect immunofluorescence studies localized seawi to the cortex of unfertilized eggs within granule-like particles, the mitotic s...
Mutations in the APC or β-catenin genes are well-established initiators of colorectal cancer, yet... more Mutations in the APC or β-catenin genes are well-established initiators of colorectal cancer, yet modifiers that facilitate the survival and progression of nascent tumor cells are not well defined. Using genetic and pharmacologic approaches in mouse colorectal cancer and human colorectal cancer xenograft models, we show that incipient intestinal tumor cells activate CDC42, an APC-interacting small GTPase, as a crucial step in malignant progression. In the mouse, Cdc42 ablation attenuated the tumorigenicity of mutant intestinal cells carrying single APC or β-catenin mutations. Similarly, human colorectal cancer with relatively higher levels of CDC42 activity was particularly sensitive to CDC42 blockade. Mechanistic studies suggested that Cdc42 may be activated at different levels, including at the level of transcriptional activation of the stem cell-enriched Rho family exchange factor Arhgef4. Our results indicate that early-stage mutant intestinal epithelial cells must recruit the p...
One of the most spectacular motions is the generation of the acrosomal process in the Limulus spe... more One of the most spectacular motions is the generation of the acrosomal process in the Limulus sperm . On contact with the egg, the sperm generates a 60-,um-long process that literally drills its way through the jelly surrounding the egg. This irreversible reaction takes only a few seconds. We suggested earlier that this motion is driven by a change in twist of the actin filaments comprising the acrosomal process. In this paper we analyze the so-called false discharge, a reversible reaction, in which the acrosomal filament bundle extends laterally from the base of the sperm and not anteriorly from the apex . Unlike the true discharge, which is straight, the false discharge is helical. Before extension, the filament bundle is coiled about the base of the sperm. In the coil, the bundle is not smoothly bent but consists of arms (straight segments) and elbows (corners) so that the coil looks like a 14-sided polygon . The extension of the false discharge works as follows: Starting at the basal end of the bundle, the filaments change their twist which concomitantly changes the orientations of the elbows relative to each other; that is, in the coil, the elbows all lie in a common plane, but, after the change in twist, the plane of each elbow is rotated to be perpendicular to that of its neighbors . This change transforms the bundle from a compact coil into an extended left-handed helix. Because the basal end of the bundle is unconstrained, the extension is lateral . The true discharge works the same way but starts at the apical end of the bundle . The apical end, however, is constrained by its passage through the nuclear canal, which directs the extension anteriorly . Unlike the false discharge, during the true discharge the elbows are melted out, making the reaction irreversible . This study shows that rapid movement can be generated by actin without myosin and gives us insight into the molecular mechanism.
Fertilization of sea urchin eggs results in the rapid polymerization of actin filaments and subse... more Fertilization of sea urchin eggs results in the rapid polymerization of actin filaments and subsequent formation of a brush border-like cortical cytoskeleton. A 110 × 10(3) Mr (110K) actin binding protein has been purified from extracts of unfertilized Strongylocentrotus purpuratus eggs. Analysis of polymerization kinetics using fluorescence and viscometry assays demonstrated that 110K accelerated the nucleation phase of actin assembly only in the presence of elevated Ca2+. The Ca(2+)-mediated effects were correlated with a decrease in sedimentable polymer and a decrease in average filament length. Addition of Ca2+ to solutions of 110K and F-actin, polymerized in the presence of EGTA, resulted in a precipitous drop in viscosity and the decreased viscosity was fully reversible upon chelation of Ca2+. The Ca2+ threshold for 110K activation was in the 10(−6) to 10(−7) M range. Nucleated assembly experiments using Limulus sperm acrosomal processes demonstrated that egg 110K capped the b...
Individual human sperm can be micromanipulated in three dimensions using a 1.06 microns Nd:YAG la... more Individual human sperm can be micromanipulated in three dimensions using a 1.06 microns Nd:YAG laser trap. Single sperm swimming with velocities in the range of 65 to 85 microns/sec can be trapped with 40 mW of power through 120 seconds without a deleterious effect on velocity. Even though it will be necessary to further evaluate the effects of laser light on specific functions of sperm, our data suggest that decreasing the time of manipulation to a minimum will increase the safety of the micromanipulation procedures. Laser traps may play a role in assisted reproductive technology by facilitating the selective transport of individual sperm.
Sea urchin coelomocytes undergo an inducible structural transformation from petalloid to filopodi... more Sea urchin coelomocytes undergo an inducible structural transformation from petalloid to filopodial form during the 'clotting' response in sea urchins. Using a petalloid coelomocyte model, stimulated coelomocytes exhibited bidirectional particle/vesicle motility with a broad distribution of velocities, ranging from 0.02 to 0.12 microns s-1 in the outward bound direction. Coelomocytes treated with the microtubule-disrupting drug, nocodazole, continued to exhibit outward particle/vesicle movements along linear paths with an average velocity of 0.028 +/- 0.006 microns s-1. We partially purified a 110 kDa polypeptide possessing K+EDTA-, Ca2(+)-, Mg2(+)- and F-actin-activated Mg(2+)-ATPase activities characteristic of myosin-like motor proteins. The 110 kDa protein immuno-crossreacted with both affinity-purified, anti-brush border unconventional myosin-I polyclonal antibodies and anti-Acanthamoeba myosin head monoclonal antibodies. By indirect immunofluorescence, the 110 kDa unco...
The motile behavior of epithelial cells located at the edge of a large wound in a monolayer of cu... more The motile behavior of epithelial cells located at the edge of a large wound in a monolayer of cultured cells was analyzed. The initial cellular response is alignment of the edge with an accompanying formation of tangential marginal actin bundles within individual cells positioned along the wound edge. Later, coherent out-growths of cell masses occur by the formation of special ''leader'' cells at the tops of outgrowths and ''follower'' cells along the sides. Leader cells exhibit profound cytoskeletal reorganization, including disassembly of marginal bundles, the realignment of actin filament bundles, and penetration of microtubules into highly active lamellae. Additionally, cell-cell contacts acquire radial geometry indicative of increased contractile tension. Interestingly, leader cells acquire a cytoskeletal organization and motility typical of fibroblasts. IAR-2 cultures stably transfected with a dominant-negative mutant of RhoA or treated with Rho-kinase inhibitor Y-27632 transformed most edge cells into leader-like cells. Alternatively , transfection of cells with constitutively active RhoA suppressed formation of leaders. Thus, expansion of the epithelial sheet involves functional differentiation into two distinct types of edge cells. The transition between these two patterns is controlled by Rho activity, which in turn controls the dynamic distribution and activity of actin filament bundles, myosin II, and microtubules.
We have investigated the Ca2+ -dependent interactions of villin, a protein of the intestinal micr... more We have investigated the Ca2+ -dependent interactions of villin, a protein of the intestinal microvillar core, with actin by monitoring resonance energy transfer between fluorescently labeled actin subunits. In the presence of elevated free Ca2+ (approximately 20 microM), villin affects both the nucleation and the elongation phases of actin polymerization. Consistent with previous reports, villin stimulates the nucleation process and will form stable nuclei under depolymerization conditions. Compared to the control, the net rate of polymerization is slightly inhibited at low concentrations of villin (villin/actin approximately 1:400) but is stimulated at higher concentrations (villin/actin greater than 1:100). Villin also significantly increases the critical concentration of actin polymerization. Addition of either villin or villin-actin complexes induces depolymerization of preassembled actin filaments. This villin-induced depolymerization is reversible upon removal of free Ca2+ or...
We used Limulus sperm acrosomal actin bundles to examine the effect of 2 uM cytochalasin B (CB) o... more We used Limulus sperm acrosomal actin bundles to examine the effect of 2 uM cytochalasin B (CB) on elongation from both the barbed and pointed ends of the actin filament. In this paper we report that 2 uM CB does not prevent monomer addition onto the barbed ends of the acrosomal actin filaments. Barbed end assembly occurred over a range of actin monomer concentrations (0.2-6 uM) in solutions conraining 75 mM KCI, 5 mM MgC12, 10 mM Imidazole, pH 7.2, and 2 uM CB. However, the elongation rates were reduced such that the rates at the barbed end were approximately the same as those at the pointed end. The association and dissociation rate constants
We have re-examined the Ca++-dependent interaction of an intestinal microvillar 95-kdalton protei... more We have re-examined the Ca++-dependent interaction of an intestinal microvillar 95-kdalton protein (MV-95K) and actin using the isolated acrosomal process bundles from Limulus sperm. Making use of the processes as nuclei for assembling actin filaments, we quantitatively and qualitatively examined MV-95K's effect on filament assembly and on Factin, both in the presence and in the absence of Ca ++. The acrosomal processes are particularly advantageous for this approach because they nucleate large numbers of filaments, they are extremely stable, and their morphology can be used to determine the polarity of any nulceated filaments. When filament nucleation was initiated in the presence of MV-95K and the absence of Ca +÷ , there was biased filament assembly from the bundle ends. The calculated elongation rates from both the barbed and pointed filament ends were virtually indistinguishable from control preparations. In the presence of Ca ÷+, MV-95K completely inhibited filament assembly from the barbed filament end without affecting the initial rate of assembly from the pointed filament end. The inhibition of assembly results from MV-95K binding to and capping the barbed filament end, thereby preventing monomer addition. This indicates that, while MV-95K is a potent nucleator of actin assembly, it is also a potent inhibitor of actin filament elongation To examine the effects of MV-95K on F-actin in the presence of Ca ++, we developed an assay where MV-95k is added to filaments previously assembled from acrosomal processes without causing filament breakage during mixing. These results clearly demonstrated that rapid filament shortening by MV-95K results through a mechanism of disrupting intrafilament monomermonomer interactions. Finally, we show that tropomyosin-containing actin filaments are insensitive to cutting, but not to capping, by MV-95K in the presence of Ca ++.
The piwi/argonaute family of proteins is involved in key developmental processes such as stem cel... more The piwi/argonaute family of proteins is involved in key developmental processes such as stem cell maintenance and axis specification through molecular mechanisms that may involve RNA silencing. Here we report on the cloning and characterization of the sea urchin piwi/argonaute family member seawi. Seawi is a major component of microtubule-ribonucleoprotein (MT-RNP) complexes isolated from two different species of sea urchin, Strongylocentrotus purpuratus and Paracentrotus lividus. Seawi co-isolates with purified ribosomes, cosediments with 80S ribosomes in sucrose density gradients, and binds microtubules. Seawi possesses the RNA binding motif common to piwi family members and binds P. lividus bep4 mRNA, a transcript that co-isolates with MT-RNP complexes and whose translation product has been shown to play a role in patterning the animal-vegetal axis. Indirect immunofluorescence studies localized seawi to the cortex of unfertilized eggs within granule-like particles, the mitotic s...
Mutations in the APC or β-catenin genes are well-established initiators of colorectal cancer, yet... more Mutations in the APC or β-catenin genes are well-established initiators of colorectal cancer, yet modifiers that facilitate the survival and progression of nascent tumor cells are not well defined. Using genetic and pharmacologic approaches in mouse colorectal cancer and human colorectal cancer xenograft models, we show that incipient intestinal tumor cells activate CDC42, an APC-interacting small GTPase, as a crucial step in malignant progression. In the mouse, Cdc42 ablation attenuated the tumorigenicity of mutant intestinal cells carrying single APC or β-catenin mutations. Similarly, human colorectal cancer with relatively higher levels of CDC42 activity was particularly sensitive to CDC42 blockade. Mechanistic studies suggested that Cdc42 may be activated at different levels, including at the level of transcriptional activation of the stem cell-enriched Rho family exchange factor Arhgef4. Our results indicate that early-stage mutant intestinal epithelial cells must recruit the p...
Sea urchin coelomocytes undergo an inducible structural transformation from petalloid to filopodi... more Sea urchin coelomocytes undergo an inducible structural transformation from petalloid to filopodial form during the 'clotting' response in sea urchins. Using a petalloid coelomocyte model, stimulated coelomocytes exhibited bidirectional particle/vesicle motility with a broad distribution of velocities, ranging from 0.02 to 0.12 microns s-1 in the outward bound direction. Coelomocytes treated with the microtubule-disrupting drug, nocodazole, continued to exhibit outward particle/vesicle movements along linear paths with an average velocity of 0.028 +/- 0.006 microns s-1. We partially purified a 110 kDa polypeptide possessing K+EDTA-, Ca2(+)-, Mg2(+)- and F-actin-activated Mg(2+)-ATPase activities characteristic of myosin-like motor proteins. The 110 kDa protein immuno-crossreacted with both affinity-purified, anti-brush border unconventional myosin-I polyclonal antibodies and anti-Acanthamoeba myosin head monoclonal antibodies. By indirect immunofluorescence, the 110 kDa unco...
The effect of villin on the critical concentration of actin and on the kinetics of its polymeriza... more The effect of villin on the critical concentration of actin and on the kinetics of its polymerization has been measured. In the presence of villin and 10 microM calcium, the critical concentration of actin increased from 0.2 to 0.9 microM. This effect of villin on the critical concentration was shown to be the result of its well-documented ability to block the "barbed" end of actin filaments, i.e., the "high-affinity end" of a polymer with a different monomer binding constant at each end. Thus, below 0.8 microM actin polymerization was prevented when the ratio of villin to actin was about 1 in 1000. Furthermore, the effect of villin was saturable; i.e., the critical concentration remained constant with increasing villin concentration once the maximal change had been obtained. In addition, fragmentation of actin filaments previously capped with villin, producing uncapped filaments, caused a rapid, transient fall of the monomer concentration. With the disappearance of the uncapped filaments the actin monomer concentration returned to that measured before fragmentation. The binding constant of villin to the barbed end of the actin filament was calculated to be greater than 10(11) M-1. The rate constants of elongation and of depolymerization at each end of an actin filament were measured. The depolymerization rate constant from the barbed end was about 10 times greater under conditions leading to complete depolymerization than under steady-state conditions. We discuss a possible explanation for the finding and its implication for possible regulatory mechanisms.
"Cutting" of actin filaments by villin was evaluated from the time course of fi... more "Cutting" of actin filaments by villin was evaluated from the time course of filament depolymerization. Depolymerization was initiated by diluting polymerized actin, labeled with a fluorescent probe on either lysine-374 or cysteine-375, to a concentration well below the critical into a medium containing free villin and various concentrations of calcium (in addition to potassium and magnesium). It was observed that at high calcium concentrations (200 microM) the time course of depolymerization could not be described by the single exponential that defines it at low calcium and low villin levels. Instead, at high calcium, the exponent increased with time and the rate of depolymerization became greater than that of controls in the absence of villin. This contrasts with the inhibition of depolymerization by villin at low calcium. The latter inhibition is a consequence of the capping of the barbed filament end by villin as are the inhibition of filament elongation and the elevation of the critical concentration. Evidence is presented that the effects of villin at high calcium are the result of cutting of the actin filaments by villin. It thus appears that different calcium binding sites control capping and cutting and that the calcium binding sites regulating cutting have a much lower affinity for calcium than the sites regulating capping of the barbed filament ends.
Communication between stem and niche supporting cells maintains the homeostasis of adult tissues.... more Communication between stem and niche supporting cells maintains the homeostasis of adult tissues. Wnt signaling is a crucial regulator of the stem cell niche, but the mechanism that governs Wnt ligand delivery in this compartment has not been fully investigated. We identified that Wnt secretion is partly dependent on Rab8a-mediated anterograde transport of Gpr177 (wntless), a Wnt-specific transmembrane transporter. Gpr177 binds to Rab8a, depletion of which compromises Gpr177 traffic, thereby weakening the secretion of multiple Wnts. Analyses of generic Wnt/β-catenin targets in Rab8a knockout mouse intestinal crypts indicate reduced signaling activities; maturation of Paneth cells - a Wnt-dependent cell type - is severely affected. Rab8a knockout crypts show an expansion of Lgr5(+) and Hopx(+) cells in vivo. However, in vitro, the knockout enteroids exhibit significantly weakened growth that can be partly restored by exogenous Wnts or Gsk3β inhibitors. Immunogold labeling and surface...
The actin cytoskeleton is an integral component of the cell-cell adherens junction complex. We us... more The actin cytoskeleton is an integral component of the cell-cell adherens junction complex. We used fluorescence labeling of actin filaments and time-lapse laser scanning confocal microscopy to investigate the functional relationship between the organization of the actin cytoskeleton and formation of adherens junctions in live epithelial cells. Rhodamine-phalloidin was loaded into cultured cells by wounding epithelial monolayers in the presence of fluorescent analog. Rhodamine-phalloidin was incorporated into the actin filaments in stress fibers, circumferential bundles, and marginal bundles. Cells containing labeled actin filaments appeared physiologically normal since the rates of migration, rates of pseudopodial protrusion/retraction, ability to form contacts, and sensitivity to cytochalasin B were equivalent to non-loaded, control epithelial cells. Marginal actin bundles initially formed as bow-shaped bundles that were observed to straighten as the bundles flowed rearward and aw...
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Papers by E. Bonder