Jemima Burden - Academia.edu (original) (raw)

Papers by Jemima Burden

arXiv (Cornell University), Feb 3, 2023

Images are at the core of most modern biological experiments and are used as a major source of qu... more Images are at the core of most modern biological experiments and are used as a major source of quantitative information. Numerous algorithms are available to process images and make them more amenable to be measured. Yet the nature of the quantitative output that is useful for a given biological experiment is uniquely dependent upon the question being investigated. Here, we discuss the 3 main types of visual information that can be extracted from microscopy data: intensity, morphology, and object counts or categorical labels. For each, we describe where they come from, how they can be measured, and what may affect the relevance of these measurements in downstream data analysis. Acknowledging that what makes a measurement "good" is ultimately down to the biological question being investigated, this review aims at providing readers with a toolkit to challenge how they quantify their own data and be critical of conclusions drawn from quantitative bioimage analysis experiments.

Journal of Thrombosis and Haemostasis, Apr 1, 2016

Essentials Deficiencies in size, number or shape of platelet granules are associated with bleedin... more Essentials Deficiencies in size, number or shape of platelet granules are associated with bleeding symptoms. Super-resolution microscopy (SRM) facilitates the diagnosis of structural platelet disorders. SRM can deliver quantitative, automated, unbiased high-throughput morphometric analyses. Using CD63 as a marker, Hermansky-Pudlak patients are easily distinguished from controls.

Molecular Biology of the Cell, Sep 1, 2010

Many proteins are retrieved to the trans-Golgi Network (TGN) from the endosomal system through se... more Many proteins are retrieved to the trans-Golgi Network (TGN) from the endosomal system through several retrograde transport pathways to maintain the composition and function of the TGN. However, the molecular mechanisms involved in these distinct retrograde pathways remain to be fully understood. Here we have used fluorescence and electron microscopy as well as various functional transport assays to show that Rab11a/b and its binding protein FIP1/RCP are both required for the retrograde delivery of TGN38 and Shiga toxin from early/recycling endosomes to the TGN, but not for the retrieval of mannose-6-phosphate receptor from late endosomes. Furthermore, by proteomic analysis we identified Golgin-97 as a FIP1/RCP-binding protein. The FIP1/RCP-binding domain maps to the C-terminus of Golgin-97, adjacent to its GRIP domain. Binding of FIP1/RCP to Golgin-97 does not affect Golgin-97 recruitment to the TGN, but appears to regulate the targeting of retrograde transport vesicles to the TGN. Thus, we propose that FIP1/RCP binding to Golgin-97 is required for tethering and fusion of recycling endosome-derived retrograde transport vesicles to the TGN.

Methods in Cell Biology, 2023

Blood, Jul 9, 2015

• Hematologic effects in the mouse model for ARC syndrome, Vps33b fl/fl-ER T2 , in which Vps33b i... more • Hematologic effects in the mouse model for ARC syndrome, Vps33b fl/fl-ER T2 , in which Vps33b is ubiquitously excised post-development. • The VPS33B-VIPAR complex is responsible for sorting cargo to and maturation of a-granule-destined MVBs. Arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome is caused by deficiencies in the trafficking proteins VPS33B or VIPAR, and is associated with a bleeding diathesis and a marked reduction in platelet a-granules. We generated a tamoxifen-inducible mouse model of VPS33B deficiency, Vps33b fl/fl-ER T2 , and studied the platelet phenotype and a-granule biogenesis. Ultrastructural analysis of Vps33b fl/fl-ER T2 platelets identified a marked reduction in a-granule count and the presence of small granule-like structures in agreement with the platelet phenotype observed in ARC patients. A reduction of ∼65% to 75% was observed in the a-granule proteins von Willebrand factor and P-selectin. Although platelet aggregation responses were not affected, a defect in d-granule secretion was observed. Under arteriolar shear conditions, Vps33b fl/fl-ER T2 platelets were unable to form stable aggregates, and tailbleeding measurement revealed a bleeding diathesis. Analysis of bone marrow-derived megakaryocytes (MKs) by conventional and immuno-electron microscopy from Vps33b fl/fl-ER T2 mice revealed a reduction in mature type-II multivesicular bodies (MVB II) and an accumulation of large vacuoles. Proteins that are normally stored in a-granules were underrepresented in MVB II and proplatelet extensions. These results demonstrate that abnormal protein trafficking and impairment in MVB maturation in MKs underlie the a-granule deficiency in Vps33b fl/fl-ER T2 mouse and ARC patients.

Methods in Enzymology, 2008

Proper development of all multicellular organisms involves programmed apoptosis. Completion of th... more Proper development of all multicellular organisms involves programmed apoptosis. Completion of this process requires removal of the resulting cell corpses through phagocytosis by their neighbors or by macrophages. Studies in C. elegans have been fruitful in the genetic dissection of key pathways, but they lack the professional immune system of higher organisms. Mammalian studies have identified a plethora of factors that are required for engulfment, but redundancy in the pathways has made it difficult to explain the genetic hierarchy of these factors. Thus, Drosophila has proven to be a useful evolutionary intermediate in which to examine this phenomenon. Here we describe methods used for dissecting the mechanisms and pathways involved in the engulfment of apoptotic cells by Drosophila phagocytes. Included are methods to be used for in vivo studies in the early embryo that can be used to examine engulfment of dying cells at various stages of embryogenesis. We also describe in vitro techniques for the use of Drosophila cell culture, including cell engulfment assays, that can be used for general phenotypic analysis, as well as live cell studies. We provide advice on imaging, including the preparation of samples for high-resolution microscopy and quantification of potential engulfment phenotypes for both in vivo and in vitro methods.

The Journal of Physiology, Mar 1, 2012

• Presynaptic terminals in hippocampal neurons are characterized by two functionally defined vesi... more • Presynaptic terminals in hippocampal neurons are characterized by two functionally defined vesicle populations: a recycling pool, which supports activity-evoked neurotransmission, and a resting pool. • Between individual synapses, the relative proportions of these two pools are highly variable, suggesting that this parameter might be specifically regulated to support changes in synaptic efficacy. • Using fluorescence imaging and correlative ultrastructural approaches we show here that a form of synaptic potentiation dependent on N-methyl-D-aspartic acid (NMDA) receptor activity can lead to a rapid and sustained expansion of the recycling fraction at the expense of the resting pool. • This recruitment of vesicles depends on nitric oxide signalling and calcineurin activity, and is accompanied by an increase in synaptic release probability. • We suggest that vesicle exchange between these pools provides a rapid mechanism to support adjustments in synaptic strength associated with a form of Hebbian plasticity.

Nature microbiology, Jul 8, 2019

To achieve efficient binding and subsequent fusion most enveloped viruses encode between 1 and 5 ... more To achieve efficient binding and subsequent fusion most enveloped viruses encode between 1 and 5 proteins 1. For many viruses, the clustering of fusion proteins, and their distribution on virus particles, is crucial for fusion activity 2,3. Poxviruses, the most complex mammalian viruses, dedicate 15 proteins to binding and membrane fusion 4. However, their spatial organization and how this influences fusion activity has not been elucidated. Here we show that the vaccinia virus membrane is organized into distinct functional domains which are critical for membrane fusion efficiency. Using super-resolution microscopy and singleparticle analysis we found that the vaccinia fusion machinery resides exclusively in clusters at virion tips. Repression of individual fusion complex components disrupts fusion machinery polarization, consistent with the reported loss of fusion activity 5. Furthermore, we show that displacement of functional fusion complexes from virion tips disrupts fusion pore formation and infection kinetics. Our results demonstrate how the protein architecture of poxviruses directly contributes to membrane fusion efficiency and suggest that nanoscale organization may be an intrinsic property of these viruses to assure successful infection.

Immunity, Oct 1, 2007

Many cells die by apoptosis during animal development. Apoptotic cells are rapidly removed throug... more Many cells die by apoptosis during animal development. Apoptotic cells are rapidly removed through phagocytosis by their neighbors or by macrophages. To genetically dissect this process, we performed an in vivo screen for genes required for efficient phagocytosis of apoptotic cells by Drosophila macrophages and identified pallbearer (pall), which encodes an F box protein. F box proteins generally provide substrate specificity to Skp Cullin F box (SCF) complexes, acting as E3 ligases that target phosphorylated proteins to ubiquitylation and degradation via the 26S proteasome. We showed that Pallbearer functions in an SCF-dependent manner and provided direct evidence of a role for ubiquitylation and proteasomal degradation in phagocytosis of apoptotic corpses in vivo. This work might further our understanding of the regulation of apoptotic cell engulfment and thus our understanding of innate immunity as a whole.

Developmental Cell, Nov 1, 2019

Highlights d Drosophila wing discs grow with spatial and temporal heterogeneity d This differenti... more Highlights d Drosophila wing discs grow with spatial and temporal heterogeneity d This differential growth determines the positions of epithelial folds d Constriction from the basement membrane is necessary for correct fold initiation d Our computational model correctly predicts the shape of growth mutants

Nature Communications, Sep 27, 2019

Mitochondrial Rho (Miro) GTPases localize to the outer mitochondrial membrane and are essential m... more Mitochondrial Rho (Miro) GTPases localize to the outer mitochondrial membrane and are essential machinery for the regulated trafficking of mitochondria to defined subcellular locations. However, their sub-mitochondrial localization and relationship with other critical mitochondrial complexes remains poorly understood. Here, using super-resolution fluorescence microscopy, we report that Miro proteins form nanometer-sized clusters along the mitochondrial outer membrane in association with the Mitochondrial Contact Site and Cristae Organizing System (MICOS). Using knockout mouse embryonic fibroblasts we show that Miro1 and Miro2 are required for normal mitochondrial cristae architecture and Endoplasmic Reticulum-Mitochondria Contacts Sites (ERMCS). Further, we show that Miro couples MICOS to TRAK motor protein adaptors to ensure the concerted transport of the two mitochondrial membranes and the correct distribution of cristae on the mitochondrial membrane. The Miro nanoscale organization, association with MICOS complex and regulation of ERMCS reveal new levels of control of the Miro GTPases on mitochondrial functionality.

Journal of Microscopy, Jun 9, 2023

Images are at the core of most modern biological experiments and are used as a major source of qu... more Images are at the core of most modern biological experiments and are used as a major source of quantitative information. Numerous algorithms are available to process images and make them more amenable to be measured. Yet the nature of the quantitative output that is useful for a given biological experiment is uniquely dependent upon the question being investigated. Here, we discuss the 3 main types of information that can be extracted from microscopy data: intensity, morphology, and object counts or categorical labels. For each, we describe where they come from, how they can be measured, and what may affect the relevance of these measurements in downstream data analysis. Acknowledging that what makes a measurement ‘good’ is ultimately down to the biological question being investigated, this review aims at providing readers with a toolkit to challenge how they quantify their own data and be critical of conclusions drawn from quantitative bioimage analysis experiments.

Journal of Visualized Experiments, Jun 9, 2022

Transmission electron microscopy has been long considered to be the gold standard for the visuali... more Transmission electron microscopy has been long considered to be the gold standard for the visualization of cellular ultrastructure. However, analysis is often limited to two dimensions, hampering the ability to fully describe the three-dimensional (3D) ultrastructure and functional relationship between organelles. Volume electron microscopy (vEM) describes a collection of techniques that enable the interrogation of cellular ultrastructure in 3D at mesoscale, microscale, and nanoscale resolutions. This protocol provides an accessible and robust method to acquire vEM data using serial section transmission EM (TEM) and covers the technical aspects of sample processing through to digital 3D reconstruction in a single, straightforward workflow. To demonstrate the usefulness of this technique, the 3D ultrastructural relationship between the endoplasmic reticulum and mitochondria and their contact sites in liver hepatocytes is presented. Interorganelle contacts serve vital roles in the transfer of ions, lipids, nutrients, and other small molecules between organelles. However, despite their initial discovery in hepatocytes, there is still much to learn about their physical features, dynamics, and functions. Interorganelle contacts can display a range of morphologies, varying in the proximity of the two organelles to one another (typically ~10-30 nm) and the extent of the contact site (from punctate contacts to larger 3D cisternal-like contacts). The examination of close contacts requires high-resolution imaging, and serial section TEM is well suited to visualize the 3D ultrastructural of interorganelle contacts during hepatocyte differentiation, as well as alterations in hepatocyte architecture associated with metabolic diseases.

Atherosclerosis, Jul 1, 2000

Developmental Cell, Feb 1, 2023

Nature Methods

Volume electron microscopy (vEM) is a group of techniques that reveal the 3D ultrastructure of ce... more Volume electron microscopy (vEM) is a group of techniques that reveal the 3D ultrastructure of cells and tissues through continuous depths of at least 1 micrometer. A burgeoning grassroots community effort is fast building the profile and revealing the impact of vEM technology in the life sciences and clinical research. The awarding of three Nobel Prizes for imaging technologies between 2008 and 2017 has highlighted the key importance of imaging in present-day life science research. The expression of proteins tagged with green fluorescent protein in living cells and organisms transformed the way in which life science research was conducted. The ability to visualize the dynamic nature of proteins in cells and tissues was recognized with the Nobel Prize in Chemistry in 2008. This was followed by another Nobel Prize in Chemistry in 2014 for the development of super-resolution light microscopy technologies, in which Abbe's resolution limit was finally broken to allow localization of fluorescently tagged molecules with a precision of tens of nanometers. Most recently, the 'resolution revolution' in cryogenic electron microscopy (cryo-EM) has enabled the determination of the molecular structure of isolated proteins and protein complexes, and was recognized with the 2017 Nobel Prize in Chemistry. There is now another imaging revolution underway that reveals the exquisite complexity of cells and tissues at the membrane and organelle scales in three dimensions-volume electron microscopy (vEM), which was recently highlighted as one of the 'seven technologies to watch in 2023' by Nature.

Nature Neuroscience

Alzheimer’s disease (AD) is characterized by synaptic loss, which can result from dysfunctional m... more Alzheimer’s disease (AD) is characterized by synaptic loss, which can result from dysfunctional microglial phagocytosis and complement activation. However, what signals drive aberrant microglia-mediated engulfment of synapses in AD is unclear. Here we report that secreted phosphoprotein 1 (SPP1/osteopontin) is upregulated predominantly by perivascular macrophages and, to a lesser extent, by perivascular fibroblasts. Perivascular SPP1 is required for microglia to engulf synapses and upregulate phagocytic markers including C1qa, Grn and Ctsb in presence of amyloid-β oligomers. Absence of Spp1 expression in AD mouse models results in prevention of synaptic loss. Furthermore, single-cell RNA sequencing and putative cell–cell interaction analyses reveal that perivascular SPP1 induces microglial phagocytic states in the hippocampus of a mouse model of AD. Altogether, we suggest a functional role for SPP1 in perivascular cells-to-microglia crosstalk, whereby SPP1 modulates microglia-mediat...

Cell shape remodeling is a principal driver of epithelial tissue morphogenesis. While progress co... more Cell shape remodeling is a principal driver of epithelial tissue morphogenesis. While progress continues to be made in our understanding of the pathways that control the apical (top) geometry of epithelial cells, we know comparatively little about those that control cell basal (bottom) geometry. To examine this issue, we used the fly ommatidium, which is the basic visual unit of the compound eye. The ommatidium is shaped as a hexagonal prism, and generating this three-dimensional structure requires ommatidial cells to adopt specific apical and basal polygonal geometries. Using this model system, we find that generating cell type-specific basal geometries starts with patterning of the basement membrane, whereby Laminin accumulates at discrete locations across the basal surface of the retina. We show that the Dystroglycan surface receptor promotes this localized Laminin accumulation. Moreover, our results reveal that localized accumulation of Laminin-Dystroglycan induces polarization ...

Virus binding serves to define virus tropism and species specificity1. Virus binding proteins are... more Virus binding serves to define virus tropism and species specificity1. Virus binding proteins are classically considered as facilitators of cell surface attachment prior to receptor engagement and virus internalization. For efficient entry vaccinia virus (VACV) – the prototypic poxvirus - relies on four binding proteins and an eleven-protein entry fusion complex (EFC)2. We recently demonstrated that VACV binding and fusion proteins are organized into distinct functional domains, with localization of EFC proteins to virion tips directly influencing membrane fusion activity3. However, the relationship between virus binding protein distribution, virion binding orientation and subsequent membrane fusion remain unexplored. Here, we show that virus binding proteins guide side-on virion binding and promote curvature of the host membrane towards EFC-containing virion tips to facilitate virus fusion. Using a cell-derived membrane-bleb model system together with super-resolution and electron ...

arXiv (Cornell University), Feb 3, 2023

Images are at the core of most modern biological experiments and are used as a major source of qu... more Images are at the core of most modern biological experiments and are used as a major source of quantitative information. Numerous algorithms are available to process images and make them more amenable to be measured. Yet the nature of the quantitative output that is useful for a given biological experiment is uniquely dependent upon the question being investigated. Here, we discuss the 3 main types of visual information that can be extracted from microscopy data: intensity, morphology, and object counts or categorical labels. For each, we describe where they come from, how they can be measured, and what may affect the relevance of these measurements in downstream data analysis. Acknowledging that what makes a measurement "good" is ultimately down to the biological question being investigated, this review aims at providing readers with a toolkit to challenge how they quantify their own data and be critical of conclusions drawn from quantitative bioimage analysis experiments.

Journal of Thrombosis and Haemostasis, Apr 1, 2016

Essentials Deficiencies in size, number or shape of platelet granules are associated with bleedin... more Essentials Deficiencies in size, number or shape of platelet granules are associated with bleeding symptoms. Super-resolution microscopy (SRM) facilitates the diagnosis of structural platelet disorders. SRM can deliver quantitative, automated, unbiased high-throughput morphometric analyses. Using CD63 as a marker, Hermansky-Pudlak patients are easily distinguished from controls.

Molecular Biology of the Cell, Sep 1, 2010

Many proteins are retrieved to the trans-Golgi Network (TGN) from the endosomal system through se... more Many proteins are retrieved to the trans-Golgi Network (TGN) from the endosomal system through several retrograde transport pathways to maintain the composition and function of the TGN. However, the molecular mechanisms involved in these distinct retrograde pathways remain to be fully understood. Here we have used fluorescence and electron microscopy as well as various functional transport assays to show that Rab11a/b and its binding protein FIP1/RCP are both required for the retrograde delivery of TGN38 and Shiga toxin from early/recycling endosomes to the TGN, but not for the retrieval of mannose-6-phosphate receptor from late endosomes. Furthermore, by proteomic analysis we identified Golgin-97 as a FIP1/RCP-binding protein. The FIP1/RCP-binding domain maps to the C-terminus of Golgin-97, adjacent to its GRIP domain. Binding of FIP1/RCP to Golgin-97 does not affect Golgin-97 recruitment to the TGN, but appears to regulate the targeting of retrograde transport vesicles to the TGN. Thus, we propose that FIP1/RCP binding to Golgin-97 is required for tethering and fusion of recycling endosome-derived retrograde transport vesicles to the TGN.

Methods in Cell Biology, 2023

Blood, Jul 9, 2015

• Hematologic effects in the mouse model for ARC syndrome, Vps33b fl/fl-ER T2 , in which Vps33b i... more • Hematologic effects in the mouse model for ARC syndrome, Vps33b fl/fl-ER T2 , in which Vps33b is ubiquitously excised post-development. • The VPS33B-VIPAR complex is responsible for sorting cargo to and maturation of a-granule-destined MVBs. Arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome is caused by deficiencies in the trafficking proteins VPS33B or VIPAR, and is associated with a bleeding diathesis and a marked reduction in platelet a-granules. We generated a tamoxifen-inducible mouse model of VPS33B deficiency, Vps33b fl/fl-ER T2 , and studied the platelet phenotype and a-granule biogenesis. Ultrastructural analysis of Vps33b fl/fl-ER T2 platelets identified a marked reduction in a-granule count and the presence of small granule-like structures in agreement with the platelet phenotype observed in ARC patients. A reduction of ∼65% to 75% was observed in the a-granule proteins von Willebrand factor and P-selectin. Although platelet aggregation responses were not affected, a defect in d-granule secretion was observed. Under arteriolar shear conditions, Vps33b fl/fl-ER T2 platelets were unable to form stable aggregates, and tailbleeding measurement revealed a bleeding diathesis. Analysis of bone marrow-derived megakaryocytes (MKs) by conventional and immuno-electron microscopy from Vps33b fl/fl-ER T2 mice revealed a reduction in mature type-II multivesicular bodies (MVB II) and an accumulation of large vacuoles. Proteins that are normally stored in a-granules were underrepresented in MVB II and proplatelet extensions. These results demonstrate that abnormal protein trafficking and impairment in MVB maturation in MKs underlie the a-granule deficiency in Vps33b fl/fl-ER T2 mouse and ARC patients.

Methods in Enzymology, 2008

Proper development of all multicellular organisms involves programmed apoptosis. Completion of th... more Proper development of all multicellular organisms involves programmed apoptosis. Completion of this process requires removal of the resulting cell corpses through phagocytosis by their neighbors or by macrophages. Studies in C. elegans have been fruitful in the genetic dissection of key pathways, but they lack the professional immune system of higher organisms. Mammalian studies have identified a plethora of factors that are required for engulfment, but redundancy in the pathways has made it difficult to explain the genetic hierarchy of these factors. Thus, Drosophila has proven to be a useful evolutionary intermediate in which to examine this phenomenon. Here we describe methods used for dissecting the mechanisms and pathways involved in the engulfment of apoptotic cells by Drosophila phagocytes. Included are methods to be used for in vivo studies in the early embryo that can be used to examine engulfment of dying cells at various stages of embryogenesis. We also describe in vitro techniques for the use of Drosophila cell culture, including cell engulfment assays, that can be used for general phenotypic analysis, as well as live cell studies. We provide advice on imaging, including the preparation of samples for high-resolution microscopy and quantification of potential engulfment phenotypes for both in vivo and in vitro methods.

The Journal of Physiology, Mar 1, 2012

• Presynaptic terminals in hippocampal neurons are characterized by two functionally defined vesi... more • Presynaptic terminals in hippocampal neurons are characterized by two functionally defined vesicle populations: a recycling pool, which supports activity-evoked neurotransmission, and a resting pool. • Between individual synapses, the relative proportions of these two pools are highly variable, suggesting that this parameter might be specifically regulated to support changes in synaptic efficacy. • Using fluorescence imaging and correlative ultrastructural approaches we show here that a form of synaptic potentiation dependent on N-methyl-D-aspartic acid (NMDA) receptor activity can lead to a rapid and sustained expansion of the recycling fraction at the expense of the resting pool. • This recruitment of vesicles depends on nitric oxide signalling and calcineurin activity, and is accompanied by an increase in synaptic release probability. • We suggest that vesicle exchange between these pools provides a rapid mechanism to support adjustments in synaptic strength associated with a form of Hebbian plasticity.

Nature microbiology, Jul 8, 2019

To achieve efficient binding and subsequent fusion most enveloped viruses encode between 1 and 5 ... more To achieve efficient binding and subsequent fusion most enveloped viruses encode between 1 and 5 proteins 1. For many viruses, the clustering of fusion proteins, and their distribution on virus particles, is crucial for fusion activity 2,3. Poxviruses, the most complex mammalian viruses, dedicate 15 proteins to binding and membrane fusion 4. However, their spatial organization and how this influences fusion activity has not been elucidated. Here we show that the vaccinia virus membrane is organized into distinct functional domains which are critical for membrane fusion efficiency. Using super-resolution microscopy and singleparticle analysis we found that the vaccinia fusion machinery resides exclusively in clusters at virion tips. Repression of individual fusion complex components disrupts fusion machinery polarization, consistent with the reported loss of fusion activity 5. Furthermore, we show that displacement of functional fusion complexes from virion tips disrupts fusion pore formation and infection kinetics. Our results demonstrate how the protein architecture of poxviruses directly contributes to membrane fusion efficiency and suggest that nanoscale organization may be an intrinsic property of these viruses to assure successful infection.

Immunity, Oct 1, 2007

Many cells die by apoptosis during animal development. Apoptotic cells are rapidly removed throug... more Many cells die by apoptosis during animal development. Apoptotic cells are rapidly removed through phagocytosis by their neighbors or by macrophages. To genetically dissect this process, we performed an in vivo screen for genes required for efficient phagocytosis of apoptotic cells by Drosophila macrophages and identified pallbearer (pall), which encodes an F box protein. F box proteins generally provide substrate specificity to Skp Cullin F box (SCF) complexes, acting as E3 ligases that target phosphorylated proteins to ubiquitylation and degradation via the 26S proteasome. We showed that Pallbearer functions in an SCF-dependent manner and provided direct evidence of a role for ubiquitylation and proteasomal degradation in phagocytosis of apoptotic corpses in vivo. This work might further our understanding of the regulation of apoptotic cell engulfment and thus our understanding of innate immunity as a whole.

Developmental Cell, Nov 1, 2019

Highlights d Drosophila wing discs grow with spatial and temporal heterogeneity d This differenti... more Highlights d Drosophila wing discs grow with spatial and temporal heterogeneity d This differential growth determines the positions of epithelial folds d Constriction from the basement membrane is necessary for correct fold initiation d Our computational model correctly predicts the shape of growth mutants

Nature Communications, Sep 27, 2019

Mitochondrial Rho (Miro) GTPases localize to the outer mitochondrial membrane and are essential m... more Mitochondrial Rho (Miro) GTPases localize to the outer mitochondrial membrane and are essential machinery for the regulated trafficking of mitochondria to defined subcellular locations. However, their sub-mitochondrial localization and relationship with other critical mitochondrial complexes remains poorly understood. Here, using super-resolution fluorescence microscopy, we report that Miro proteins form nanometer-sized clusters along the mitochondrial outer membrane in association with the Mitochondrial Contact Site and Cristae Organizing System (MICOS). Using knockout mouse embryonic fibroblasts we show that Miro1 and Miro2 are required for normal mitochondrial cristae architecture and Endoplasmic Reticulum-Mitochondria Contacts Sites (ERMCS). Further, we show that Miro couples MICOS to TRAK motor protein adaptors to ensure the concerted transport of the two mitochondrial membranes and the correct distribution of cristae on the mitochondrial membrane. The Miro nanoscale organization, association with MICOS complex and regulation of ERMCS reveal new levels of control of the Miro GTPases on mitochondrial functionality.

Journal of Microscopy, Jun 9, 2023

Images are at the core of most modern biological experiments and are used as a major source of qu... more Images are at the core of most modern biological experiments and are used as a major source of quantitative information. Numerous algorithms are available to process images and make them more amenable to be measured. Yet the nature of the quantitative output that is useful for a given biological experiment is uniquely dependent upon the question being investigated. Here, we discuss the 3 main types of information that can be extracted from microscopy data: intensity, morphology, and object counts or categorical labels. For each, we describe where they come from, how they can be measured, and what may affect the relevance of these measurements in downstream data analysis. Acknowledging that what makes a measurement ‘good’ is ultimately down to the biological question being investigated, this review aims at providing readers with a toolkit to challenge how they quantify their own data and be critical of conclusions drawn from quantitative bioimage analysis experiments.

Journal of Visualized Experiments, Jun 9, 2022

Transmission electron microscopy has been long considered to be the gold standard for the visuali... more Transmission electron microscopy has been long considered to be the gold standard for the visualization of cellular ultrastructure. However, analysis is often limited to two dimensions, hampering the ability to fully describe the three-dimensional (3D) ultrastructure and functional relationship between organelles. Volume electron microscopy (vEM) describes a collection of techniques that enable the interrogation of cellular ultrastructure in 3D at mesoscale, microscale, and nanoscale resolutions. This protocol provides an accessible and robust method to acquire vEM data using serial section transmission EM (TEM) and covers the technical aspects of sample processing through to digital 3D reconstruction in a single, straightforward workflow. To demonstrate the usefulness of this technique, the 3D ultrastructural relationship between the endoplasmic reticulum and mitochondria and their contact sites in liver hepatocytes is presented. Interorganelle contacts serve vital roles in the transfer of ions, lipids, nutrients, and other small molecules between organelles. However, despite their initial discovery in hepatocytes, there is still much to learn about their physical features, dynamics, and functions. Interorganelle contacts can display a range of morphologies, varying in the proximity of the two organelles to one another (typically ~10-30 nm) and the extent of the contact site (from punctate contacts to larger 3D cisternal-like contacts). The examination of close contacts requires high-resolution imaging, and serial section TEM is well suited to visualize the 3D ultrastructural of interorganelle contacts during hepatocyte differentiation, as well as alterations in hepatocyte architecture associated with metabolic diseases.

Atherosclerosis, Jul 1, 2000

Developmental Cell, Feb 1, 2023

Nature Methods

Volume electron microscopy (vEM) is a group of techniques that reveal the 3D ultrastructure of ce... more Volume electron microscopy (vEM) is a group of techniques that reveal the 3D ultrastructure of cells and tissues through continuous depths of at least 1 micrometer. A burgeoning grassroots community effort is fast building the profile and revealing the impact of vEM technology in the life sciences and clinical research. The awarding of three Nobel Prizes for imaging technologies between 2008 and 2017 has highlighted the key importance of imaging in present-day life science research. The expression of proteins tagged with green fluorescent protein in living cells and organisms transformed the way in which life science research was conducted. The ability to visualize the dynamic nature of proteins in cells and tissues was recognized with the Nobel Prize in Chemistry in 2008. This was followed by another Nobel Prize in Chemistry in 2014 for the development of super-resolution light microscopy technologies, in which Abbe's resolution limit was finally broken to allow localization of fluorescently tagged molecules with a precision of tens of nanometers. Most recently, the 'resolution revolution' in cryogenic electron microscopy (cryo-EM) has enabled the determination of the molecular structure of isolated proteins and protein complexes, and was recognized with the 2017 Nobel Prize in Chemistry. There is now another imaging revolution underway that reveals the exquisite complexity of cells and tissues at the membrane and organelle scales in three dimensions-volume electron microscopy (vEM), which was recently highlighted as one of the 'seven technologies to watch in 2023' by Nature.

Nature Neuroscience

Alzheimer’s disease (AD) is characterized by synaptic loss, which can result from dysfunctional m... more Alzheimer’s disease (AD) is characterized by synaptic loss, which can result from dysfunctional microglial phagocytosis and complement activation. However, what signals drive aberrant microglia-mediated engulfment of synapses in AD is unclear. Here we report that secreted phosphoprotein 1 (SPP1/osteopontin) is upregulated predominantly by perivascular macrophages and, to a lesser extent, by perivascular fibroblasts. Perivascular SPP1 is required for microglia to engulf synapses and upregulate phagocytic markers including C1qa, Grn and Ctsb in presence of amyloid-β oligomers. Absence of Spp1 expression in AD mouse models results in prevention of synaptic loss. Furthermore, single-cell RNA sequencing and putative cell–cell interaction analyses reveal that perivascular SPP1 induces microglial phagocytic states in the hippocampus of a mouse model of AD. Altogether, we suggest a functional role for SPP1 in perivascular cells-to-microglia crosstalk, whereby SPP1 modulates microglia-mediat...

Cell shape remodeling is a principal driver of epithelial tissue morphogenesis. While progress co... more Cell shape remodeling is a principal driver of epithelial tissue morphogenesis. While progress continues to be made in our understanding of the pathways that control the apical (top) geometry of epithelial cells, we know comparatively little about those that control cell basal (bottom) geometry. To examine this issue, we used the fly ommatidium, which is the basic visual unit of the compound eye. The ommatidium is shaped as a hexagonal prism, and generating this three-dimensional structure requires ommatidial cells to adopt specific apical and basal polygonal geometries. Using this model system, we find that generating cell type-specific basal geometries starts with patterning of the basement membrane, whereby Laminin accumulates at discrete locations across the basal surface of the retina. We show that the Dystroglycan surface receptor promotes this localized Laminin accumulation. Moreover, our results reveal that localized accumulation of Laminin-Dystroglycan induces polarization ...

Virus binding serves to define virus tropism and species specificity1. Virus binding proteins are... more Virus binding serves to define virus tropism and species specificity1. Virus binding proteins are classically considered as facilitators of cell surface attachment prior to receptor engagement and virus internalization. For efficient entry vaccinia virus (VACV) – the prototypic poxvirus - relies on four binding proteins and an eleven-protein entry fusion complex (EFC)2. We recently demonstrated that VACV binding and fusion proteins are organized into distinct functional domains, with localization of EFC proteins to virion tips directly influencing membrane fusion activity3. However, the relationship between virus binding protein distribution, virion binding orientation and subsequent membrane fusion remain unexplored. Here, we show that virus binding proteins guide side-on virion binding and promote curvature of the host membrane towards EFC-containing virion tips to facilitate virus fusion. Using a cell-derived membrane-bleb model system together with super-resolution and electron ...