Heidi Hehnly | Syracuse University (original) (raw)

Papers by Heidi Hehnly

Scientific Reports, 2016

The geometry of the cleavage furrow during mitosis is often asymmetric in vivo and plays a critic... more The geometry of the cleavage furrow during mitosis is often asymmetric in vivo and plays a critical role in stem cell differentiation and the relative positioning of daughter cells during development. Early observations of adhesive cell lines revealed asymmetry in the shape of the cleavage furrow, where the bottom (i.e., substrate attached side) of the cleavage furrow ingressed less than the top (i.e., unattached side). This data suggested substrate attachment could be regulating furrow ingression. Here we report a population of mitotic focal adhesions (FAs) controls the symmetry of the cleavage furrow. In single HeLa cells, stronger adhesion to the substrate directed less ingression from the bottom of the cell through a pathway including paxillin, focal adhesion kinase (FAK) and vinculin. Cell-cell contacts also direct ingression of the cleavage furrow in coordination with FAs in epithelial cells-MDCK-within monolayers and polarized cysts. In addition, mitotic FAs established 3D orientation of the mitotic spindle and the relative positioning of mother and daughter centrosomes. Therefore, our data reveals mitotic FAs as a key link between mitotic cell shape and spindle orientation, and may have important implications in our understanding stem cell homeostasis and tumorigenesis.

Scientific Reports, 2016

The geometry of the cleavage furrow during mitosis is often asymmetric in vivo and plays a critic... more The geometry of the cleavage furrow during mitosis is often asymmetric in vivo and plays a critical role in stem cell differentiation and the relative positioning of daughter cells during development. Early observations of adhesive cell lines revealed asymmetry in the shape of the cleavage furrow, where the bottom (i.e., substrate attached side) of the cleavage furrow ingressed less than the top (i.e., unattached side). This data suggested substrate attachment could be regulating furrow ingression. Here we report a population of mitotic focal adhesions (FAs) controls the symmetry of the cleavage furrow. In single HeLa cells, stronger adhesion to the substrate directed less ingression from the bottom of the cell through a pathway including paxillin, focal adhesion kinase (FAK) and vinculin. Cell-cell contacts also direct ingression of the cleavage furrow in coordination with FAs in epithelial cells-MDCK-within monolayers and polarized cysts. In addition, mitotic FAs established 3D orientation of the mitotic spindle and the relative positioning of mother and daughter centrosomes. Therefore, our data reveals mitotic FAs as a key link between mitotic cell shape and spindle orientation, and may have important implications in our understanding stem cell homeostasis and tumorigenesis.

Current Biology, 2014

Majewski osteodysplastic primordial dwarfism type II (MOP-DII) is caused by mutations in the cent... more Majewski osteodysplastic primordial dwarfism type II (MOP-DII) is caused by mutations in the centrosome gene pericentrin (PCNT) that lead to severe pre-and postnatal growth retardation . As in MOPDII patients, disruption of pericentrin (Pcnt) in mice caused a number of abnormalities including microcephaly, aberrant hemodynamics analyzed by in utero echocardiography, and cardiovascular anomalies; the latter being associated with mortality, as in the human condition . To identify the mechanisms underlying these defects, we tested for changes in cell and molecular function. All Pcnt 2/2 mouse tissues and cells examined showed spindle misorientation. This mouse phenotype was associated with misdirected ventricular septal growth in the heart, decreased proliferative symmetric divisions in brain neural progenitors, and increased misoriented divisions in fibroblasts; the same phenotype was seen in fibroblasts from three MOPDII individuals. Misoriented spindles were associated with disrupted astral microtubules and near complete loss of a unique set of centrosome proteins from spindle poles (ninein, Cep215, centriolin). All these proteins appear to be crucial for microtubule anchoring and all interacted with Pcnt, suggesting that Pcnt serves as a molecular scaffold for this functionally linked set of spindle pole proteins. Importantly, Pcnt disruption had no detectable effect on localization of proteins involved in the cortical polarity pathway (NuMA, p150 glued , aPKC). Not only do these data reveal a spindle-pole-localized complex for spindle orientation, but they identify key spindle symmetry proteins involved in the pathogenesis of MOPDII.

Molecular Biology of the Cell, 2012

Cell polarity is important for a number of processes, from chemotaxis to embryogenesis. Recent st... more Cell polarity is important for a number of processes, from chemotaxis to embryogenesis. Recent studies suggest a new role for polarity in the orchestration of events during the final cell separation step of cell division called abscission. Abscission shares several features with cell polarization, including rearrangement of phosphatidylinositols, reorganization of microtubules, and trafficking of exocyst-associated membranes. Here we focus on how the canonical pathways for cell polarization and cell migration may play a role in spatiotemporal membrane trafficking events required for the final stages of cytokinesis.

ACS chemical biology, Jan 13, 2015

A-Kinase Anchoring Proteins (AKAPs) coordinate complex signaling events by serving as spatiotempo... more A-Kinase Anchoring Proteins (AKAPs) coordinate complex signaling events by serving as spatiotemporal modulators of cAMP-dependent Protein Kinase activity in cells. Although AKAPs organize a plethora of diverse pathways, their cellular roles are often elusive due to the dynamic nature of these signaling complexes. AKAPs can interact with the type I or type II PKA holoenzymes by virtue of high affinity interactions with the R subunits. As a means to delineate AKAP-mediated PKA signaling in cells, we sought to develop isoform-selective disruptors of AKAP signaling. Here we report the development of conformationally constrained peptides named RI-STapled Anchoring Disruptors (RI-STADs) that target the docking/dimerization domain of the type 1 regulatory subunit of PKA. These high affinity peptides are isoform-selective for the RI isoforms, can outcompete binding by the classical AKAP disruptor Ht31 and selectively displace RIα, but not RIIα, from binding the dual-specific AKAP149 complex. Importantly, these peptides are cell-permeable and disrupt Type I PKA-mediated phosphorylation events in the context of live cells. Hence, RI-STAD peptides are versatile cellular tools to selectively probe anchored type I PKA signaling events.

eLife, 2014

Virus-host interactions drive a remarkable diversity of immune responses and countermeasures. We ... more Virus-host interactions drive a remarkable diversity of immune responses and countermeasures. We found that two RNA viruses with broad host ranges, vesicular stomatitis virus (VSV) and Sindbis virus (SINV), are completely restricted in their replication after entry into Lepidopteran cells. This restriction is overcome when cells are co-infected with vaccinia virus (VACV), a vertebrate DNA virus. Using RNAi screening, we show that Lepidopteran RNAi, Nuclear Factor-κB, and ubiquitin-proteasome pathways restrict RNA virus infection. Surprisingly, a highly conserved, uncharacterized VACV protein, A51R, can partially overcome this virus restriction. We show that A51R is also critical for VACV replication in vertebrate cells and for pathogenesis in mice. Interestingly, A51R colocalizes with, and stabilizes, host microtubules and also associates with ubiquitin. We show that A51R promotes viral protein stability, possibly by preventing ubiquitindependent targeting of viral proteins for destruction. Importantly, our studies reveal exciting new opportunities to study virus-host interactions in experimentally-tractable Lepidopteran systems.

Current biology : CB, Jan 6, 2014

Majewski osteodysplastic primordial dwarfism type II (MOP-DII) is caused by mutations in the cent... more Majewski osteodysplastic primordial dwarfism type II (MOP-DII) is caused by mutations in the centrosome gene pericentrin (PCNT) that lead to severe pre-and postnatal growth retardation . As in MOPDII patients, disruption of pericentrin (Pcnt) in mice caused a number of abnormalities including microcephaly, aberrant hemodynamics analyzed by in utero echocardiography, and cardiovascular anomalies; the latter being associated with mortality, as in the human condition . To identify the mechanisms underlying these defects, we tested for changes in cell and molecular function. All Pcnt 2/2 mouse tissues and cells examined showed spindle misorientation. This mouse phenotype was associated with misdirected ventricular septal growth in the heart, decreased proliferative symmetric divisions in brain neural progenitors, and increased misoriented divisions in fibroblasts; the same phenotype was seen in fibroblasts from three MOPDII individuals. Misoriented spindles were associated with disrupted astral microtubules and near complete loss of a unique set of centrosome proteins from spindle poles (ninein, Cep215, centriolin). All these proteins appear to be crucial for microtubule anchoring and all interacted with Pcnt, suggesting that Pcnt serves as a molecular scaffold for this functionally linked set of spindle pole proteins. Importantly, Pcnt disruption had no detectable effect on localization of proteins involved in the cortical polarity pathway (NuMA, p150 glued , aPKC). Not only do these data reveal a spindle-pole-localized complex for spindle orientation, but they identify key spindle symmetry proteins involved in the pathogenesis of MOPDII.

Small GTPases, 2014

A recent study revealed new roles for the Rab11 GTPase during mitosis. Rab11 is involved in recyc... more A recent study revealed new roles for the Rab11 GTPase during mitosis. Rab11 is involved in recycling endosome localization to mitotic spindle poles via dynein-mediated transport. This process is in contrast to Golgi membranes, which disperse in mitosis and do not appear to directly contribute to mitotic functions. Rab11-depletion prevents recycling endosome organization at spindle poles, delays mitotic progression, and disrupts spindle pole protein recruitment, astral microtubule organization, and mitotic spindle orientation. However, Rab11 is not the only endocytic and/or trafficking protein that regulates mitotic progression. Clathrin and two small GTPases (Rab6A', Rab5) play key roles in spindle organization and function. In this commentary, we discuss the roles of all these canonical endocytic and membrane trafficking proteins during mitosis and speculate on possible crosscommunication between them and their molecular pathways that ensure faithful progression through mitosis.

Current Biology, 2014

Majewski osteodysplastic primordial dwarfism type II (MOP-DII) is caused by mutations in the cent... more Majewski osteodysplastic primordial dwarfism type II (MOP-DII) is caused by mutations in the centrosome gene pericentrin (PCNT) that lead to severe pre-and postnatal growth retardation . As in MOPDII patients, disruption of pericentrin (Pcnt) in mice caused a number of abnormalities including microcephaly, aberrant hemodynamics analyzed by in utero echocardiography, and cardiovascular anomalies; the latter being associated with mortality, as in the human condition . To identify the mechanisms underlying these defects, we tested for changes in cell and molecular function. All Pcnt 2/2 mouse tissues and cells examined showed spindle misorientation. This mouse phenotype was associated with misdirected ventricular septal growth in the heart, decreased proliferative symmetric divisions in brain neural progenitors, and increased misoriented divisions in fibroblasts; the same phenotype was seen in fibroblasts from three MOPDII individuals. Misoriented spindles were associated with disrupted astral microtubules and near complete loss of a unique set of centrosome proteins from spindle poles (ninein, Cep215, centriolin). All these proteins appear to be crucial for microtubule anchoring and all interacted with Pcnt, suggesting that Pcnt serves as a molecular scaffold for this functionally linked set of spindle pole proteins. Importantly, Pcnt disruption had no detectable effect on localization of proteins involved in the cortical polarity pathway (NuMA, p150 glued , aPKC). Not only do these data reveal a spindle-pole-localized complex for spindle orientation, but they identify key spindle symmetry proteins involved in the pathogenesis of MOPDII.

Traffic, 2010

The molecular mechanisms underlying cytoskeletondependent Golgi positioning are poorly understood... more The molecular mechanisms underlying cytoskeletondependent Golgi positioning are poorly understood. In mammalian cells, the Golgi apparatus is localized near the juxtanuclear centrosome via dynein-mediated motility along microtubules. Previous studies implicate Cdc42 in regulating dynein-dependent motility. Here we show that reduced expression of the Cdc42-specific GTPaseactivating protein, ARHGAP21, inhibits the ability of dispersed Golgi membranes to reposition at the centrosome following nocodazole treatment and washout. Cdc42 regulation of Golgi positioning appears to involve ARF1 and a binding interaction with the vesicle-coat protein coatomer. We tested whether Cdc42 directly affects motility, as opposed to the formation of a trafficking intermediate, using a Golgi capture and motility assay in permeabilized cells. Disrupting Cdc42 activation or the coatomer/Cdc42 binding interaction stimulated Golgi motility. The coatomer/Cdc42-sensitive motility was blocked by the addition of an inhibitory dynein antibody. Together, our results reveal that dynein and microtubuledependent Golgi positioning is regulated by ARF1-, coatomer-, and ARHGAP21-dependent Cdc42 signaling.

Molecular Biology of the Cell, 2006

The bacterial exotoxin Shiga toxin is endocytosed by mammalian host cells and transported retrogr... more The bacterial exotoxin Shiga toxin is endocytosed by mammalian host cells and transported retrogradely through the secretory pathway before entering the cytosol. Shiga toxin also increases the levels of microfilaments and microtubules (MTs) upon binding to the cell surface. The purpose for this alteration in cytoskeletal dynamics is unknown. We have investigated whether Shiga toxin-induced changes in MT levels facilitate its intracellular transport. We have tested the effects of the Shiga toxin B subunit (STB) on MT-dependent and -independent transport steps. STB increases the rate of MT-dependent Golgi stack repositioning after nocodazole treatment. It also enhances the MT-dependent accumulation of transferrin in a perinuclear recycling compartment. By contrast, the rate of MT-independent transferrin recycling is not significantly different when STB is present. We found that STB normally requires MTs and dynein for its retrograde transport to the juxtanuclear Golgi complex and that STB increases MT assembly. Furthermore, we find that MT polymerization is limiting for STB transport in cells. These results show that STB-induced changes in cytoskeletal dynamics influence intracellular transport. We conclude that the increased rate of MT assembly upon Shiga toxin binding facilitates the retrograde transport of the toxin through the secretory pathway.

FEBS Letters, 2007

During vesicular transport, the assembly of the coat complexes and the selection of cargo protein... more During vesicular transport, the assembly of the coat complexes and the selection of cargo proteins must be coordinated with the subsequent translocation of vesicles from the donor to an acceptor compartment. Here, we review recent progress toward uncovering the molecular mechanisms that connect transport vesicles to the protein machinery responsible for cytoskeleton-mediated motility. An emerging theme is that vesicle cargo proteins, either directly or through binding interactions with coat proteins, are able to influence cytoskeletal dynamics and motor protein function. Hence, a vesicle's cargo composition may help direct its intracellular motility and targeting.

Developmental Cell, 2014

During interphase, Rab11-GTPase-containing endosomes recycle endocytic cargo. However, little is ... more During interphase, Rab11-GTPase-containing endosomes recycle endocytic cargo. However, little is known about Rab11 endosomes in mitosis. Here, we show that Rab11 localizes to the mitotic spindle and regulates dynein-dependent endosome localization at poles. We found that mitotic recycling endosomes bind g-TuRC components and associate with tubulin in vitro. Rab11 depletion or dominantnegative Rab11 expression disrupts astral microtubules, delays mitosis, and redistributes spindle pole proteins. Reciprocally, constitutively active Rab11 increases astral microtubules, restores g-tubulin spindle pole localization, and generates robust spindles. This suggests a role for Rab11 activity in spindle pole maturation during mitosis. Rab11 depletion causes misorientation of the mitotic spindle and the plane of cell division. These findings suggest a molecular mechanism for the organization of astral microtubules and the mitotic spindle through Rab11dependent control of spindle pole assembly and function. We propose that Rab11 and its associated endosomes cocontribute to these processes through retrograde transport to poles by dynein.

Current Biology, 2012

The recycling endosome localizes to a pericentrosomal region via microtubule-dependent transport.... more The recycling endosome localizes to a pericentrosomal region via microtubule-dependent transport. We previously showed that Sec15, an effector of the recycling endosome component, Rab11-GTPase, interacts with the mother centriole appendage protein, centriolin, suggesting an interaction between endosomes and centrosomes . Here we show that the recycling endosome associates with the appendages of the mother (older) centriole. We show that two mother centriole appendage proteins, centriolin and cenexin/ODF2, regulate association of the endosome components Rab11, the Rab11 GTP-activating protein Evi5, and the exocyst at the mother centriole. Development of an in vitro method for reconstituting endosome protein complexes onto isolated membrane-free centrosomes demonstrates that purified GTP-Rab11 but not GDP-Rab11 binds to mother centriole appendages in the absence of membranes. Moreover, centriolin depletion displaces the centrosomal Rab11 GAP, Evi5, and increases mothercentriole-associated Rab11; depletion of Evi5 also increases centrosomal Rab11. This indicates that centriolin localizes Evi5 to centriolar appendages to turn off centrosomal Rab11 activity. Finally, centriolin depletion disrupts recycling endosome organization and function, suggesting a role for mother centriole proteins in the regulation of Rab11 localization and activity at the mother centriole.

Cell Cycle, 2013

and roger.bouillon@med.kuleuven.be; http: //dx.doi.org/10.4161/cc.24306 Already in the eighties i... more and roger.bouillon@med.kuleuven.be; http: //dx.doi.org/10.4161/cc.24306 Already in the eighties it was shown that the active form of vitamin D 3 , 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ], can inhibit the proliferation of melanoma cells 1 and stimulate the differentiation of myeloid leukemia cells. 2 The almost universal presence of the vitamin D receptor (VDR) and the presence of 1α-hydroxylase (CyP27B1) activity in non-classical tissues together with the antiproliferative and prodifferentiating effects suggests a paracrine role for 1,25(OH) 2 D 3 .

Molecular biology of the …, 2009

Shiga-toxin-producing Escherichia coli remain a food-borne health threat. Shiga toxin is endocyto... more Shiga-toxin-producing Escherichia coli remain a food-borne health threat. Shiga toxin is endocytosed by intestinal epithelial cells and transported retrogradely through the secretory pathway. It is ultimately translocated to the cytosol where it inhibits protein translation. We found that Shiga toxin transport through the secretory pathway was dependent on the cytoskeleton. Recent studies reveal that Shiga toxin activates signaling pathways that affect microtubule reassembly and dynein-dependent motility. We propose that Shiga toxin alters cytoskeletal dynamics in a way that facilitates its transport through the secretory pathway. We have now found that Rho GTPases regulate the endocytosis and retrograde motility of Shiga toxin. The expression of RhoA mutants inhibited endocytosis of Shiga toxin. Constitutively active Cdc42 or knockdown of the Cdc42specific GAP, ARHGAP21, inhibited the transport of Shiga toxin to the juxtanuclear Golgi apparatus. The ability of Shiga toxin to stimulate microtubule-based transferrin transport also required Cdc42 and ARHGAP21 function. Shiga toxin addition greatly decreases the levels of active Cdc42-GTP in an ARHGAP21-dependent manner. We conclude that ARHGAP21 and Cdc42-based signaling regulates the dynein-dependent retrograde transport of Shiga toxin to the Golgi apparatus. ‡ Present address:

Cell Cycle, 2015

The mitotic spindle apparatus is composed of microtubule (MT) networks attached to kinetochores o... more The mitotic spindle apparatus is composed of microtubule (MT) networks attached to kinetochores organized from 2 centrosomes (a.k.a. spindle poles). In addition to this central spindle apparatus, astral MTs assemble at the mitotic spindle pole and attach to the cell cortex to ensure appropriate spindle orientation. We propose that cell cycle-related kinase, Nek7, and its novel interacting protein RGS2, are involved in mitosis regulation and spindle formation. We found that RGS2 localizes to the mitotic spindle in a Nek7-dependent manner, and along with Nek7 contributes to spindle morphology and mitotic spindle pole integrity. RGS2-depletion leads to a mitotic-delay and severe defects in the chromosomes alignment and congression. Importantly, RGS2 or Nek7 depletion or even overexpression of wild-type or kinase-dead Nek7, reduced g-tubulin from the mitotic spindle poles. In addition to causing a mitotic delay, RGS2 depletion induced mitotic spindle misorientation coinciding with astral MT-reduction. We propose that these phenotypes directly contribute to a failure in mitotic spindle alignment to the substratum. In conclusion, we suggest a molecular mechanism whereupon Nek7 and RGS2 may act cooperatively to ensure proper mitotic spindle organization.

Scientific Reports, 2016

The geometry of the cleavage furrow during mitosis is often asymmetric in vivo and plays a critic... more The geometry of the cleavage furrow during mitosis is often asymmetric in vivo and plays a critical role in stem cell differentiation and the relative positioning of daughter cells during development. Early observations of adhesive cell lines revealed asymmetry in the shape of the cleavage furrow, where the bottom (i.e., substrate attached side) of the cleavage furrow ingressed less than the top (i.e., unattached side). This data suggested substrate attachment could be regulating furrow ingression. Here we report a population of mitotic focal adhesions (FAs) controls the symmetry of the cleavage furrow. In single HeLa cells, stronger adhesion to the substrate directed less ingression from the bottom of the cell through a pathway including paxillin, focal adhesion kinase (FAK) and vinculin. Cell-cell contacts also direct ingression of the cleavage furrow in coordination with FAs in epithelial cells-MDCK-within monolayers and polarized cysts. In addition, mitotic FAs established 3D orientation of the mitotic spindle and the relative positioning of mother and daughter centrosomes. Therefore, our data reveals mitotic FAs as a key link between mitotic cell shape and spindle orientation, and may have important implications in our understanding stem cell homeostasis and tumorigenesis.

Scientific Reports, 2016

The geometry of the cleavage furrow during mitosis is often asymmetric in vivo and plays a critic... more The geometry of the cleavage furrow during mitosis is often asymmetric in vivo and plays a critical role in stem cell differentiation and the relative positioning of daughter cells during development. Early observations of adhesive cell lines revealed asymmetry in the shape of the cleavage furrow, where the bottom (i.e., substrate attached side) of the cleavage furrow ingressed less than the top (i.e., unattached side). This data suggested substrate attachment could be regulating furrow ingression. Here we report a population of mitotic focal adhesions (FAs) controls the symmetry of the cleavage furrow. In single HeLa cells, stronger adhesion to the substrate directed less ingression from the bottom of the cell through a pathway including paxillin, focal adhesion kinase (FAK) and vinculin. Cell-cell contacts also direct ingression of the cleavage furrow in coordination with FAs in epithelial cells-MDCK-within monolayers and polarized cysts. In addition, mitotic FAs established 3D orientation of the mitotic spindle and the relative positioning of mother and daughter centrosomes. Therefore, our data reveals mitotic FAs as a key link between mitotic cell shape and spindle orientation, and may have important implications in our understanding stem cell homeostasis and tumorigenesis.

Current Biology, 2014

Majewski osteodysplastic primordial dwarfism type II (MOP-DII) is caused by mutations in the cent... more Majewski osteodysplastic primordial dwarfism type II (MOP-DII) is caused by mutations in the centrosome gene pericentrin (PCNT) that lead to severe pre-and postnatal growth retardation . As in MOPDII patients, disruption of pericentrin (Pcnt) in mice caused a number of abnormalities including microcephaly, aberrant hemodynamics analyzed by in utero echocardiography, and cardiovascular anomalies; the latter being associated with mortality, as in the human condition . To identify the mechanisms underlying these defects, we tested for changes in cell and molecular function. All Pcnt 2/2 mouse tissues and cells examined showed spindle misorientation. This mouse phenotype was associated with misdirected ventricular septal growth in the heart, decreased proliferative symmetric divisions in brain neural progenitors, and increased misoriented divisions in fibroblasts; the same phenotype was seen in fibroblasts from three MOPDII individuals. Misoriented spindles were associated with disrupted astral microtubules and near complete loss of a unique set of centrosome proteins from spindle poles (ninein, Cep215, centriolin). All these proteins appear to be crucial for microtubule anchoring and all interacted with Pcnt, suggesting that Pcnt serves as a molecular scaffold for this functionally linked set of spindle pole proteins. Importantly, Pcnt disruption had no detectable effect on localization of proteins involved in the cortical polarity pathway (NuMA, p150 glued , aPKC). Not only do these data reveal a spindle-pole-localized complex for spindle orientation, but they identify key spindle symmetry proteins involved in the pathogenesis of MOPDII.

Molecular Biology of the Cell, 2012

Cell polarity is important for a number of processes, from chemotaxis to embryogenesis. Recent st... more Cell polarity is important for a number of processes, from chemotaxis to embryogenesis. Recent studies suggest a new role for polarity in the orchestration of events during the final cell separation step of cell division called abscission. Abscission shares several features with cell polarization, including rearrangement of phosphatidylinositols, reorganization of microtubules, and trafficking of exocyst-associated membranes. Here we focus on how the canonical pathways for cell polarization and cell migration may play a role in spatiotemporal membrane trafficking events required for the final stages of cytokinesis.

ACS chemical biology, Jan 13, 2015

A-Kinase Anchoring Proteins (AKAPs) coordinate complex signaling events by serving as spatiotempo... more A-Kinase Anchoring Proteins (AKAPs) coordinate complex signaling events by serving as spatiotemporal modulators of cAMP-dependent Protein Kinase activity in cells. Although AKAPs organize a plethora of diverse pathways, their cellular roles are often elusive due to the dynamic nature of these signaling complexes. AKAPs can interact with the type I or type II PKA holoenzymes by virtue of high affinity interactions with the R subunits. As a means to delineate AKAP-mediated PKA signaling in cells, we sought to develop isoform-selective disruptors of AKAP signaling. Here we report the development of conformationally constrained peptides named RI-STapled Anchoring Disruptors (RI-STADs) that target the docking/dimerization domain of the type 1 regulatory subunit of PKA. These high affinity peptides are isoform-selective for the RI isoforms, can outcompete binding by the classical AKAP disruptor Ht31 and selectively displace RIα, but not RIIα, from binding the dual-specific AKAP149 complex. Importantly, these peptides are cell-permeable and disrupt Type I PKA-mediated phosphorylation events in the context of live cells. Hence, RI-STAD peptides are versatile cellular tools to selectively probe anchored type I PKA signaling events.

eLife, 2014

Virus-host interactions drive a remarkable diversity of immune responses and countermeasures. We ... more Virus-host interactions drive a remarkable diversity of immune responses and countermeasures. We found that two RNA viruses with broad host ranges, vesicular stomatitis virus (VSV) and Sindbis virus (SINV), are completely restricted in their replication after entry into Lepidopteran cells. This restriction is overcome when cells are co-infected with vaccinia virus (VACV), a vertebrate DNA virus. Using RNAi screening, we show that Lepidopteran RNAi, Nuclear Factor-κB, and ubiquitin-proteasome pathways restrict RNA virus infection. Surprisingly, a highly conserved, uncharacterized VACV protein, A51R, can partially overcome this virus restriction. We show that A51R is also critical for VACV replication in vertebrate cells and for pathogenesis in mice. Interestingly, A51R colocalizes with, and stabilizes, host microtubules and also associates with ubiquitin. We show that A51R promotes viral protein stability, possibly by preventing ubiquitindependent targeting of viral proteins for destruction. Importantly, our studies reveal exciting new opportunities to study virus-host interactions in experimentally-tractable Lepidopteran systems.

Current biology : CB, Jan 6, 2014

Majewski osteodysplastic primordial dwarfism type II (MOP-DII) is caused by mutations in the cent... more Majewski osteodysplastic primordial dwarfism type II (MOP-DII) is caused by mutations in the centrosome gene pericentrin (PCNT) that lead to severe pre-and postnatal growth retardation . As in MOPDII patients, disruption of pericentrin (Pcnt) in mice caused a number of abnormalities including microcephaly, aberrant hemodynamics analyzed by in utero echocardiography, and cardiovascular anomalies; the latter being associated with mortality, as in the human condition . To identify the mechanisms underlying these defects, we tested for changes in cell and molecular function. All Pcnt 2/2 mouse tissues and cells examined showed spindle misorientation. This mouse phenotype was associated with misdirected ventricular septal growth in the heart, decreased proliferative symmetric divisions in brain neural progenitors, and increased misoriented divisions in fibroblasts; the same phenotype was seen in fibroblasts from three MOPDII individuals. Misoriented spindles were associated with disrupted astral microtubules and near complete loss of a unique set of centrosome proteins from spindle poles (ninein, Cep215, centriolin). All these proteins appear to be crucial for microtubule anchoring and all interacted with Pcnt, suggesting that Pcnt serves as a molecular scaffold for this functionally linked set of spindle pole proteins. Importantly, Pcnt disruption had no detectable effect on localization of proteins involved in the cortical polarity pathway (NuMA, p150 glued , aPKC). Not only do these data reveal a spindle-pole-localized complex for spindle orientation, but they identify key spindle symmetry proteins involved in the pathogenesis of MOPDII.

Small GTPases, 2014

A recent study revealed new roles for the Rab11 GTPase during mitosis. Rab11 is involved in recyc... more A recent study revealed new roles for the Rab11 GTPase during mitosis. Rab11 is involved in recycling endosome localization to mitotic spindle poles via dynein-mediated transport. This process is in contrast to Golgi membranes, which disperse in mitosis and do not appear to directly contribute to mitotic functions. Rab11-depletion prevents recycling endosome organization at spindle poles, delays mitotic progression, and disrupts spindle pole protein recruitment, astral microtubule organization, and mitotic spindle orientation. However, Rab11 is not the only endocytic and/or trafficking protein that regulates mitotic progression. Clathrin and two small GTPases (Rab6A', Rab5) play key roles in spindle organization and function. In this commentary, we discuss the roles of all these canonical endocytic and membrane trafficking proteins during mitosis and speculate on possible crosscommunication between them and their molecular pathways that ensure faithful progression through mitosis.

Current Biology, 2014

Majewski osteodysplastic primordial dwarfism type II (MOP-DII) is caused by mutations in the cent... more Majewski osteodysplastic primordial dwarfism type II (MOP-DII) is caused by mutations in the centrosome gene pericentrin (PCNT) that lead to severe pre-and postnatal growth retardation . As in MOPDII patients, disruption of pericentrin (Pcnt) in mice caused a number of abnormalities including microcephaly, aberrant hemodynamics analyzed by in utero echocardiography, and cardiovascular anomalies; the latter being associated with mortality, as in the human condition . To identify the mechanisms underlying these defects, we tested for changes in cell and molecular function. All Pcnt 2/2 mouse tissues and cells examined showed spindle misorientation. This mouse phenotype was associated with misdirected ventricular septal growth in the heart, decreased proliferative symmetric divisions in brain neural progenitors, and increased misoriented divisions in fibroblasts; the same phenotype was seen in fibroblasts from three MOPDII individuals. Misoriented spindles were associated with disrupted astral microtubules and near complete loss of a unique set of centrosome proteins from spindle poles (ninein, Cep215, centriolin). All these proteins appear to be crucial for microtubule anchoring and all interacted with Pcnt, suggesting that Pcnt serves as a molecular scaffold for this functionally linked set of spindle pole proteins. Importantly, Pcnt disruption had no detectable effect on localization of proteins involved in the cortical polarity pathway (NuMA, p150 glued , aPKC). Not only do these data reveal a spindle-pole-localized complex for spindle orientation, but they identify key spindle symmetry proteins involved in the pathogenesis of MOPDII.

Traffic, 2010

The molecular mechanisms underlying cytoskeletondependent Golgi positioning are poorly understood... more The molecular mechanisms underlying cytoskeletondependent Golgi positioning are poorly understood. In mammalian cells, the Golgi apparatus is localized near the juxtanuclear centrosome via dynein-mediated motility along microtubules. Previous studies implicate Cdc42 in regulating dynein-dependent motility. Here we show that reduced expression of the Cdc42-specific GTPaseactivating protein, ARHGAP21, inhibits the ability of dispersed Golgi membranes to reposition at the centrosome following nocodazole treatment and washout. Cdc42 regulation of Golgi positioning appears to involve ARF1 and a binding interaction with the vesicle-coat protein coatomer. We tested whether Cdc42 directly affects motility, as opposed to the formation of a trafficking intermediate, using a Golgi capture and motility assay in permeabilized cells. Disrupting Cdc42 activation or the coatomer/Cdc42 binding interaction stimulated Golgi motility. The coatomer/Cdc42-sensitive motility was blocked by the addition of an inhibitory dynein antibody. Together, our results reveal that dynein and microtubuledependent Golgi positioning is regulated by ARF1-, coatomer-, and ARHGAP21-dependent Cdc42 signaling.

Molecular Biology of the Cell, 2006

The bacterial exotoxin Shiga toxin is endocytosed by mammalian host cells and transported retrogr... more The bacterial exotoxin Shiga toxin is endocytosed by mammalian host cells and transported retrogradely through the secretory pathway before entering the cytosol. Shiga toxin also increases the levels of microfilaments and microtubules (MTs) upon binding to the cell surface. The purpose for this alteration in cytoskeletal dynamics is unknown. We have investigated whether Shiga toxin-induced changes in MT levels facilitate its intracellular transport. We have tested the effects of the Shiga toxin B subunit (STB) on MT-dependent and -independent transport steps. STB increases the rate of MT-dependent Golgi stack repositioning after nocodazole treatment. It also enhances the MT-dependent accumulation of transferrin in a perinuclear recycling compartment. By contrast, the rate of MT-independent transferrin recycling is not significantly different when STB is present. We found that STB normally requires MTs and dynein for its retrograde transport to the juxtanuclear Golgi complex and that STB increases MT assembly. Furthermore, we find that MT polymerization is limiting for STB transport in cells. These results show that STB-induced changes in cytoskeletal dynamics influence intracellular transport. We conclude that the increased rate of MT assembly upon Shiga toxin binding facilitates the retrograde transport of the toxin through the secretory pathway.

FEBS Letters, 2007

During vesicular transport, the assembly of the coat complexes and the selection of cargo protein... more During vesicular transport, the assembly of the coat complexes and the selection of cargo proteins must be coordinated with the subsequent translocation of vesicles from the donor to an acceptor compartment. Here, we review recent progress toward uncovering the molecular mechanisms that connect transport vesicles to the protein machinery responsible for cytoskeleton-mediated motility. An emerging theme is that vesicle cargo proteins, either directly or through binding interactions with coat proteins, are able to influence cytoskeletal dynamics and motor protein function. Hence, a vesicle's cargo composition may help direct its intracellular motility and targeting.

Developmental Cell, 2014

During interphase, Rab11-GTPase-containing endosomes recycle endocytic cargo. However, little is ... more During interphase, Rab11-GTPase-containing endosomes recycle endocytic cargo. However, little is known about Rab11 endosomes in mitosis. Here, we show that Rab11 localizes to the mitotic spindle and regulates dynein-dependent endosome localization at poles. We found that mitotic recycling endosomes bind g-TuRC components and associate with tubulin in vitro. Rab11 depletion or dominantnegative Rab11 expression disrupts astral microtubules, delays mitosis, and redistributes spindle pole proteins. Reciprocally, constitutively active Rab11 increases astral microtubules, restores g-tubulin spindle pole localization, and generates robust spindles. This suggests a role for Rab11 activity in spindle pole maturation during mitosis. Rab11 depletion causes misorientation of the mitotic spindle and the plane of cell division. These findings suggest a molecular mechanism for the organization of astral microtubules and the mitotic spindle through Rab11dependent control of spindle pole assembly and function. We propose that Rab11 and its associated endosomes cocontribute to these processes through retrograde transport to poles by dynein.

Current Biology, 2012

The recycling endosome localizes to a pericentrosomal region via microtubule-dependent transport.... more The recycling endosome localizes to a pericentrosomal region via microtubule-dependent transport. We previously showed that Sec15, an effector of the recycling endosome component, Rab11-GTPase, interacts with the mother centriole appendage protein, centriolin, suggesting an interaction between endosomes and centrosomes . Here we show that the recycling endosome associates with the appendages of the mother (older) centriole. We show that two mother centriole appendage proteins, centriolin and cenexin/ODF2, regulate association of the endosome components Rab11, the Rab11 GTP-activating protein Evi5, and the exocyst at the mother centriole. Development of an in vitro method for reconstituting endosome protein complexes onto isolated membrane-free centrosomes demonstrates that purified GTP-Rab11 but not GDP-Rab11 binds to mother centriole appendages in the absence of membranes. Moreover, centriolin depletion displaces the centrosomal Rab11 GAP, Evi5, and increases mothercentriole-associated Rab11; depletion of Evi5 also increases centrosomal Rab11. This indicates that centriolin localizes Evi5 to centriolar appendages to turn off centrosomal Rab11 activity. Finally, centriolin depletion disrupts recycling endosome organization and function, suggesting a role for mother centriole proteins in the regulation of Rab11 localization and activity at the mother centriole.

Cell Cycle, 2013

and roger.bouillon@med.kuleuven.be; http: //dx.doi.org/10.4161/cc.24306 Already in the eighties i... more and roger.bouillon@med.kuleuven.be; http: //dx.doi.org/10.4161/cc.24306 Already in the eighties it was shown that the active form of vitamin D 3 , 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ], can inhibit the proliferation of melanoma cells 1 and stimulate the differentiation of myeloid leukemia cells. 2 The almost universal presence of the vitamin D receptor (VDR) and the presence of 1α-hydroxylase (CyP27B1) activity in non-classical tissues together with the antiproliferative and prodifferentiating effects suggests a paracrine role for 1,25(OH) 2 D 3 .

Molecular biology of the …, 2009

Shiga-toxin-producing Escherichia coli remain a food-borne health threat. Shiga toxin is endocyto... more Shiga-toxin-producing Escherichia coli remain a food-borne health threat. Shiga toxin is endocytosed by intestinal epithelial cells and transported retrogradely through the secretory pathway. It is ultimately translocated to the cytosol where it inhibits protein translation. We found that Shiga toxin transport through the secretory pathway was dependent on the cytoskeleton. Recent studies reveal that Shiga toxin activates signaling pathways that affect microtubule reassembly and dynein-dependent motility. We propose that Shiga toxin alters cytoskeletal dynamics in a way that facilitates its transport through the secretory pathway. We have now found that Rho GTPases regulate the endocytosis and retrograde motility of Shiga toxin. The expression of RhoA mutants inhibited endocytosis of Shiga toxin. Constitutively active Cdc42 or knockdown of the Cdc42specific GAP, ARHGAP21, inhibited the transport of Shiga toxin to the juxtanuclear Golgi apparatus. The ability of Shiga toxin to stimulate microtubule-based transferrin transport also required Cdc42 and ARHGAP21 function. Shiga toxin addition greatly decreases the levels of active Cdc42-GTP in an ARHGAP21-dependent manner. We conclude that ARHGAP21 and Cdc42-based signaling regulates the dynein-dependent retrograde transport of Shiga toxin to the Golgi apparatus. ‡ Present address:

Cell Cycle, 2015

The mitotic spindle apparatus is composed of microtubule (MT) networks attached to kinetochores o... more The mitotic spindle apparatus is composed of microtubule (MT) networks attached to kinetochores organized from 2 centrosomes (a.k.a. spindle poles). In addition to this central spindle apparatus, astral MTs assemble at the mitotic spindle pole and attach to the cell cortex to ensure appropriate spindle orientation. We propose that cell cycle-related kinase, Nek7, and its novel interacting protein RGS2, are involved in mitosis regulation and spindle formation. We found that RGS2 localizes to the mitotic spindle in a Nek7-dependent manner, and along with Nek7 contributes to spindle morphology and mitotic spindle pole integrity. RGS2-depletion leads to a mitotic-delay and severe defects in the chromosomes alignment and congression. Importantly, RGS2 or Nek7 depletion or even overexpression of wild-type or kinase-dead Nek7, reduced g-tubulin from the mitotic spindle poles. In addition to causing a mitotic delay, RGS2 depletion induced mitotic spindle misorientation coinciding with astral MT-reduction. We propose that these phenotypes directly contribute to a failure in mitotic spindle alignment to the substratum. In conclusion, we suggest a molecular mechanism whereupon Nek7 and RGS2 may act cooperatively to ensure proper mitotic spindle organization.