SummaryPlatelets are cell fragments of various size that help maintain hemostasis. The way platel... more SummaryPlatelets are cell fragments of various size that help maintain hemostasis. The way platelets respond during a clotting process is known to depend on their size, with important physiological consequences. We characterized the cytoskeleton of platelets as a function of their size. In resting Human and Mice platelets, we find a quadradic law between the size of a platelet and the amount of microtubule polymer it contains. We further estimate the length and number of microtubules in the marginal band using Electron and Super-resolution microscopy. In platelets activated with ADP, the marginal band coils as a consequence of cortical contraction driven by actin. We observe that this elastic coiling response is accompanied by a reversible shortening of the marginal band. Moreover, larger platelets have a higher propensity to coil. These results establish the dynamic equilibrium that is responsible for platelet size and differential response on a more quantitative level.HighlightsPl...
During neurotransmission, synaptic vesicles undergo multiple rounds of exo-endocytosis, involving... more During neurotransmission, synaptic vesicles undergo multiple rounds of exo-endocytosis, involving recycling and/or degradation of synaptic proteins. While ubiquitin signaling at synapses is essential for neural function, it has been assumed that synaptic proteostasis requires the ubiquitin-proteasome system (UPS). We demonstrate here that turnover of synaptic membrane proteins via the endolysosomal pathway is essential for synaptic function. In both human and mouse, hypomorphic mutations in the ubiquitin adaptor protein PLAA cause an infantile-lethal neurodysfunction syndrome with seizures. Resulting from perturbed endolysosomal degradation, Plaa mutant neurons accumulate K63-polyubiquitylated proteins and synaptic membrane proteins, disrupting synaptic vesicle recycling and neurotransmission. Through characterization of this neurological intracellular trafficking disorder, we establish the importance of ubiquitin-mediated endolysosomal trafficking at the synapse.
Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of carg... more Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo within cilia, but how this cargo is selectively transported to cilia is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to enrich in diverse classes of ciliary membrane proteins. In Wdr35 mouse mutants, the non-core IFT-A components are degraded and core components accumulate at the ciliary base. We reveal deep sequence homology of WDR35 and other IFT-A subunits to α and ß' COPI coatomer subunits, and demonstrate an accumulation of 'coat-less' vesicles which fail to fuse with Wdr35 mutant cilia. We determine that recombinant non-core IFT-As can bind directly to lipids and provide the first in-situ evidence of a novel coat function for WDR35, likely with other IFT-A proteins, in delivering ciliary membrane cargo necessary for cilia elongation.
Centrosomes are orbited by centriolar satellites, dynamic multiprotein assemblies nucleated by PC... more Centrosomes are orbited by centriolar satellites, dynamic multiprotein assemblies nucleated by PCM1. To study the requirement for centriolar satellites, we generated mice lacking PCM1. Pcm1−/− mice display partially penetrant perinatal lethality with survivors exhibiting hydrocephalus, oligospermia and cerebellar hypoplasia, as well as variable expressivity of other ciliopathy features including cystic kidneys. Pcm1−/− multiciliated ependymal cells and PCM1−/− retinal pigmented epithelial 1 (RPE1) cells showed reduced ciliogenesis. PCM1−/− RPE1 cells displayed reduced docking of the mother centriole to the ciliary vesicle and removal of CP110 and CEP97 from the distal mother centriole, indicating compromized early ciliogenesis. We show these molecular cascades are maintained in vivo, and we suggest that the cellular threshold to trigger ciliogenesis varies between cell types. We propose that PCM1 and centriolar satellites facilitate efficient trafficking of proteins to and from cent...
Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven 12 transport of c... more Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven 12 transport of cargo within cilia, but how this cargo is selectively transported to cilia and across the 13 diffusion barrier is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for 14 its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to 15 enrich in diverse classes of ciliary membrane proteins. In Wdr35mouse mutants, the IFT-A 16 peripheral components are degraded and core components accumulate at the transition zone. 17 We reveal deep sequence homology and structural similarity of WDR35 and other IFT-As to the 18 coatomer COPI proteins and ’, and demonstrate an accumulation of ‘coat-less’ vesicles which 19 fail to fuse with Wdr35mutant cilia. Our data provides the first in situ evidence of a novel 20 coatomer function for WDR35 likely with other IFT-A proteins in delivering ciliary membrane 21 cargo from the Golgi necessary for cili...
Cilia are microtubule-based organelles present on the surface of almost all mammalian cells that ... more Cilia are microtubule-based organelles present on the surface of almost all mammalian cells that play key sensory and sometimes motile functions. Defects in cilia structure or function lead to a group of human diseases called the ciliopathies. In order to function, cilia must maintain a distinct protein and membrane composition from the surrounding plasma membrane and cytosol, highly enriched in signaling receptors and effectors. How this compartmentalization occurs remains unclear. There is no known protein synthesising machinery in cilia and the transition zone at the base of cilium forms the diffusion barrier, which does not allow free exchange between the cytoplasm to cilioplasm. Biogenesis of the cilium (ciliogenesis) requires many carriers and adaptors to facilitate passage of cargo across the transition zone. Once inside the cilia intraflagellar transport (IFT) proteins move the cargo along microtubules 'railways' of the axoneme. IFTs are known to assemble in two protein complexes, IFTA and IFTB protein complexes. IFT-B complex known to be made of 16 different proteins mediate anterograde transport with the help of kinesin motors, and IFT-A complex made of 6 different proteins help in retrograde transport powered by dynein motors. However, the exact mechanism of transport of cargo to cilia and entry across the diffusion barrier is not well understood, and the functions of each IFT protein remain unclear. In this thesis, I describe the critical role of one of the IFT-A complex protein WDR35/IFT121 in the formation of functional cilia by transporting structural elements of cilia via a vesicular mediated pathway. Null mutations in the IFT-A component WDR35/ IFT121 are embryonic lethal in both mouse models and human ciliopathies (Mill et al., 2011). Small, unstable WDR35 mutant cilia are formed but fail to become enriched in diverse classes of integral and membrane-associated proteins (Caparrós-Martín JA et al., 2015; Fu W et al., 2016). To elucidate its role in the entry of membrane proteins to cilia, I prese [...]
Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of carg... more Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo within cilia, but how this cargo is selectively transported to cilia and across the diffusion barrier is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to enrich in diverse classes of ciliary membrane proteins. In Wdr35 mouse mutants, the IFT-A peripheral components are degraded and core components accumulate at the transition zone. We reveal deep sequence homology and structural similarity of WDR35 and other IFT-As to the coatomer COPI proteins a and β′, and demonstrate an accumulation of ‘coat-less’ vesicles which fail to fuse with Wdr35 mutant cilia. Our data provides the first in situ evidence of a novel coatomer function for WDR35 likely with other IFT-A proteins in delivering ciliary membrane cargo from the Golgi necessary for cilia elongation.
We present new fluorophore-conjugates for dual-color photoactivation and super-resolution imaging... more We present new fluorophore-conjugates for dual-color photoactivation and super-resolution imaging inside live mammalian cells.
Emma A Hall, Michael S Nahorski, Lyndsay M Murray, Ranad Shaheen , Emma Perkins, Kosala N Dissana... more Emma A Hall, Michael S Nahorski, Lyndsay M Murray, Ranad Shaheen , Emma Perkins, Kosala N Dissanayake, Yosua Kristaryanto, Ross A Jones, Julie Vogt, Manon Rivagorda, Mark T Handley, Girish R Mali, Tooba Quidwai, Dinesh C Soares, Margaret A Keighren, Lisa McKie, Richard L Mort, Noor Gammoh, Amaya Garcia-Munoz, Tracey Davey, Matthieu Vermeren, Diana Walsh, Peter Budd, Irene A Aligianis, Eissa Faqeih, Alan J Quigley, Ian J Jackson, Yogesh Kulathu, Mandy Jackson, Richard R Ribchester, Alex von Kriegsheim, Fowzan S. Alkuraya, C Geoffrey Woods, Eamonn R Maher & Pleasantine Mill
Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of carg... more Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo within cilia, but how this cargo is selectively transported to cilia is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to enrich in diverse classes of ciliary membrane proteins. In Wdr35 mouse mutants, the non-core IFT-A components are degraded and core components accumulate at the ciliary base. We reveal deep sequence homology of WDR35 and other IFT-A subunits to α and ß' COPI coatomer subunits, and demonstrate an accumulation of 'coat-less' vesicles which fail to fuse with Wdr35 mutant cilia. We determine that recombinant non-core IFT-As can bind directly to lipids and provide the first in-situ evidence of a novel coat function for WDR35, likely with other IFT-A proteins, in delivering ciliary membrane cargo necessary for cilia elongation.
SummaryPlatelets are cell fragments of various size that help maintain hemostasis. The way platel... more SummaryPlatelets are cell fragments of various size that help maintain hemostasis. The way platelets respond during a clotting process is known to depend on their size, with important physiological consequences. We characterized the cytoskeleton of platelets as a function of their size. In resting Human and Mice platelets, we find a quadradic law between the size of a platelet and the amount of microtubule polymer it contains. We further estimate the length and number of microtubules in the marginal band using Electron and Super-resolution microscopy. In platelets activated with ADP, the marginal band coils as a consequence of cortical contraction driven by actin. We observe that this elastic coiling response is accompanied by a reversible shortening of the marginal band. Moreover, larger platelets have a higher propensity to coil. These results establish the dynamic equilibrium that is responsible for platelet size and differential response on a more quantitative level.HighlightsPl...
During neurotransmission, synaptic vesicles undergo multiple rounds of exo-endocytosis, involving... more During neurotransmission, synaptic vesicles undergo multiple rounds of exo-endocytosis, involving recycling and/or degradation of synaptic proteins. While ubiquitin signaling at synapses is essential for neural function, it has been assumed that synaptic proteostasis requires the ubiquitin-proteasome system (UPS). We demonstrate here that turnover of synaptic membrane proteins via the endolysosomal pathway is essential for synaptic function. In both human and mouse, hypomorphic mutations in the ubiquitin adaptor protein PLAA cause an infantile-lethal neurodysfunction syndrome with seizures. Resulting from perturbed endolysosomal degradation, Plaa mutant neurons accumulate K63-polyubiquitylated proteins and synaptic membrane proteins, disrupting synaptic vesicle recycling and neurotransmission. Through characterization of this neurological intracellular trafficking disorder, we establish the importance of ubiquitin-mediated endolysosomal trafficking at the synapse.
Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of carg... more Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo within cilia, but how this cargo is selectively transported to cilia is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to enrich in diverse classes of ciliary membrane proteins. In Wdr35 mouse mutants, the non-core IFT-A components are degraded and core components accumulate at the ciliary base. We reveal deep sequence homology of WDR35 and other IFT-A subunits to α and ß' COPI coatomer subunits, and demonstrate an accumulation of 'coat-less' vesicles which fail to fuse with Wdr35 mutant cilia. We determine that recombinant non-core IFT-As can bind directly to lipids and provide the first in-situ evidence of a novel coat function for WDR35, likely with other IFT-A proteins, in delivering ciliary membrane cargo necessary for cilia elongation.
Centrosomes are orbited by centriolar satellites, dynamic multiprotein assemblies nucleated by PC... more Centrosomes are orbited by centriolar satellites, dynamic multiprotein assemblies nucleated by PCM1. To study the requirement for centriolar satellites, we generated mice lacking PCM1. Pcm1−/− mice display partially penetrant perinatal lethality with survivors exhibiting hydrocephalus, oligospermia and cerebellar hypoplasia, as well as variable expressivity of other ciliopathy features including cystic kidneys. Pcm1−/− multiciliated ependymal cells and PCM1−/− retinal pigmented epithelial 1 (RPE1) cells showed reduced ciliogenesis. PCM1−/− RPE1 cells displayed reduced docking of the mother centriole to the ciliary vesicle and removal of CP110 and CEP97 from the distal mother centriole, indicating compromized early ciliogenesis. We show these molecular cascades are maintained in vivo, and we suggest that the cellular threshold to trigger ciliogenesis varies between cell types. We propose that PCM1 and centriolar satellites facilitate efficient trafficking of proteins to and from cent...
Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven 12 transport of c... more Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven 12 transport of cargo within cilia, but how this cargo is selectively transported to cilia and across the 13 diffusion barrier is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for 14 its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to 15 enrich in diverse classes of ciliary membrane proteins. In Wdr35mouse mutants, the IFT-A 16 peripheral components are degraded and core components accumulate at the transition zone. 17 We reveal deep sequence homology and structural similarity of WDR35 and other IFT-As to the 18 coatomer COPI proteins and ’, and demonstrate an accumulation of ‘coat-less’ vesicles which 19 fail to fuse with Wdr35mutant cilia. Our data provides the first in situ evidence of a novel 20 coatomer function for WDR35 likely with other IFT-A proteins in delivering ciliary membrane 21 cargo from the Golgi necessary for cili...
Cilia are microtubule-based organelles present on the surface of almost all mammalian cells that ... more Cilia are microtubule-based organelles present on the surface of almost all mammalian cells that play key sensory and sometimes motile functions. Defects in cilia structure or function lead to a group of human diseases called the ciliopathies. In order to function, cilia must maintain a distinct protein and membrane composition from the surrounding plasma membrane and cytosol, highly enriched in signaling receptors and effectors. How this compartmentalization occurs remains unclear. There is no known protein synthesising machinery in cilia and the transition zone at the base of cilium forms the diffusion barrier, which does not allow free exchange between the cytoplasm to cilioplasm. Biogenesis of the cilium (ciliogenesis) requires many carriers and adaptors to facilitate passage of cargo across the transition zone. Once inside the cilia intraflagellar transport (IFT) proteins move the cargo along microtubules 'railways' of the axoneme. IFTs are known to assemble in two protein complexes, IFTA and IFTB protein complexes. IFT-B complex known to be made of 16 different proteins mediate anterograde transport with the help of kinesin motors, and IFT-A complex made of 6 different proteins help in retrograde transport powered by dynein motors. However, the exact mechanism of transport of cargo to cilia and entry across the diffusion barrier is not well understood, and the functions of each IFT protein remain unclear. In this thesis, I describe the critical role of one of the IFT-A complex protein WDR35/IFT121 in the formation of functional cilia by transporting structural elements of cilia via a vesicular mediated pathway. Null mutations in the IFT-A component WDR35/ IFT121 are embryonic lethal in both mouse models and human ciliopathies (Mill et al., 2011). Small, unstable WDR35 mutant cilia are formed but fail to become enriched in diverse classes of integral and membrane-associated proteins (Caparrós-Martín JA et al., 2015; Fu W et al., 2016). To elucidate its role in the entry of membrane proteins to cilia, I prese [...]
Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of carg... more Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo within cilia, but how this cargo is selectively transported to cilia and across the diffusion barrier is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to enrich in diverse classes of ciliary membrane proteins. In Wdr35 mouse mutants, the IFT-A peripheral components are degraded and core components accumulate at the transition zone. We reveal deep sequence homology and structural similarity of WDR35 and other IFT-As to the coatomer COPI proteins a and β′, and demonstrate an accumulation of ‘coat-less’ vesicles which fail to fuse with Wdr35 mutant cilia. Our data provides the first in situ evidence of a novel coatomer function for WDR35 likely with other IFT-A proteins in delivering ciliary membrane cargo from the Golgi necessary for cilia elongation.
We present new fluorophore-conjugates for dual-color photoactivation and super-resolution imaging... more We present new fluorophore-conjugates for dual-color photoactivation and super-resolution imaging inside live mammalian cells.
Emma A Hall, Michael S Nahorski, Lyndsay M Murray, Ranad Shaheen , Emma Perkins, Kosala N Dissana... more Emma A Hall, Michael S Nahorski, Lyndsay M Murray, Ranad Shaheen , Emma Perkins, Kosala N Dissanayake, Yosua Kristaryanto, Ross A Jones, Julie Vogt, Manon Rivagorda, Mark T Handley, Girish R Mali, Tooba Quidwai, Dinesh C Soares, Margaret A Keighren, Lisa McKie, Richard L Mort, Noor Gammoh, Amaya Garcia-Munoz, Tracey Davey, Matthieu Vermeren, Diana Walsh, Peter Budd, Irene A Aligianis, Eissa Faqeih, Alan J Quigley, Ian J Jackson, Yogesh Kulathu, Mandy Jackson, Richard R Ribchester, Alex von Kriegsheim, Fowzan S. Alkuraya, C Geoffrey Woods, Eamonn R Maher & Pleasantine Mill
Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of carg... more Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo within cilia, but how this cargo is selectively transported to cilia is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to enrich in diverse classes of ciliary membrane proteins. In Wdr35 mouse mutants, the non-core IFT-A components are degraded and core components accumulate at the ciliary base. We reveal deep sequence homology of WDR35 and other IFT-A subunits to α and ß' COPI coatomer subunits, and demonstrate an accumulation of 'coat-less' vesicles which fail to fuse with Wdr35 mutant cilia. We determine that recombinant non-core IFT-As can bind directly to lipids and provide the first in-situ evidence of a novel coat function for WDR35, likely with other IFT-A proteins, in delivering ciliary membrane cargo necessary for cilia elongation.
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