@article {67177, title = {Actin cables and comet tails organize mitochondrial networks in mitosis.}, journal = {Nature}, volume = {591}, year = {2021}, month = {2021 Mar 03}, pages = {659-664}, abstract = {

Symmetric cell division requires the even partitioning of genetic information and cytoplasmic contents between daughter cells. Whereas the mechanisms coordinating the segregation of the genome are well known, the processes that ensure organelle segregation between daughter cells remain less well understood. Here we identify multiple actin assemblies with distinct but complementary roles in mitochondrial organization and inheritance in mitosis. First, we find a dense meshwork of subcortical actin cables assembled throughout the mitotic cytoplasm. This network scaffolds the endoplasmic reticulum and organizes three-dimensional mitochondrial positioning to ensure the equal segregation of mitochondrial mass at cytokinesis. Second, we identify a dynamic wave of actin filaments reversibly assembling on the surface of mitochondria during mitosis. Mitochondria sampled by this wave are enveloped within actin clouds that can spontaneously break symmetry to form elongated comet tails. Mitochondrial comet tails promote randomly directed bursts of movement that shuffle mitochondrial position within the mother cell to randomize inheritance of healthy and damaged mitochondria between daughter cells. Thus, parallel mechanisms mediated by the actin cytoskeleton ensure both equal and random inheritance of mitochondria in symmetrically dividing cells.

}, issn = {1476-4687}, doi = {10.1038/s41586-021-03309-5}, author = {Moore, Andrew S and Coscia, Stephen M and Simpson, Cory L and Ortega, Fabian E and Wait, Eric C and Heddleston, John M and Nirschl, Jeffrey J and Obara, Christopher J and Guedes-Dias, Pedro and Boecker, C Alexander and Chew, Teng-Leong and Theriot, Julie A and Lippincott-Schwartz, Jennifer and Holzbaur, Erika L F} } @article {67710, title = {Endothelial junctional membrane protrusions serve as hotspots for neutrophil transmigration.}, journal = {eLife}, volume = {10}, year = {2021}, month = {2021 Aug 25}, abstract = {

Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed tissue. Evidence accumulates that leukocytes use preferred exit sites, though it is not yet clear how these hotspots in the endothelium are defined and how they are recognized by the leukocyte. Using lattice light sheet microscopy, we discovered that leukocytes prefer endothelial membrane protrusions at cell junctions for transmigration. Phenotypically, these junctional membrane protrusions are present in an asymmetric manner, meaning that one endothelial cell shows the protrusion and the adjacent one does not. Consequently, leukocytes cross the junction by migrating underneath the protruding endothelial cell. These protrusions depend on Rac1 activity and by using a photo-activatable Rac1 probe, we could artificially generate local exit-sites for leukocytes. Overall, we have discovered a new mechanism that uses local induced junctional membrane protrusions to facilitate/steer the leukocyte escape/exit from inflamed vessel walls.

}, issn = {2050-084X}, doi = {10.7554/eLife.66074}, author = {Arts, Janine Jg and Mahlandt, Eike K and Gr{\"o}nloh, Max and Schimmel, Lilian and Noordstra, Ivar and Gordon, Emma and van Steen, Abraham Ci and Tol, Simon and Walzog, Barbara and van Rijssel, Jos and Nolte, Martijn A and Postma, Marten and Khuon, Satya and Heddleston, John M and Wait, Eric and Chew, Teng Leong and Winter, Mark and Montanez, Eloi and Goedhart, Joachim and van Buul, Jaap D} } @article {66021, title = {The level of oncogenic Ras determines the malignant transformation of Lkb1 mutant tissue in vivo.}, journal = {Communications Biology}, volume = {4}, year = {2021}, month = {2021 Jan 29}, pages = {142}, abstract = {

The genetic and metabolic heterogeneity of RAS-driven cancers has confounded therapeutic strategies in the clinic. To address this, rapid and genetically tractable animal models are needed that recapitulate the heterogeneity of RAS-driven cancers in vivo. Here, we generate a Drosophila melanogaster model of Ras/Lkb1 mutant carcinoma. We show that low-level expression of oncogenic Ras (Ras) promotes the survival of Lkb1 mutant tissue, but results in autonomous cell cycle arrest and non-autonomous overgrowth of wild-type tissue. In contrast, high-level expression of oncogenic Ras (Ras) transforms Lkb1 mutant tissue resulting in lethal malignant tumors. Using simultaneous multiview light-sheet microcopy, we have characterized invasion phenotypes of Ras/Lkb1 tumors in living larvae. Our molecular analysis reveals sustained activation of the AMPK pathway in malignant Ras/Lkb1 tumors, and demonstrate the genetic and pharmacologic dependence of these tumors on CaMK-activated Ampk. We further show that LKB1 mutant human lung adenocarcinoma patients with high levels of oncogenic KRAS exhibit worse overall survival and increased AMPK activation. Our results suggest that high levels of oncogenic KRAS is a driving event in the malignant transformation of LKB1 mutant tissue, and uncovers a vulnerability that may be used to target this aggressive genetic subset of RAS-driven tumors.

}, issn = {2399-3642}, doi = {10.1038/s42003-021-01663-8}, author = {Rackley, Briana and Seong, Chang-Soo and Kiely, Evan and Parker, Rebecca E and Rupji, Manali and Dwivedi, Bhakti and Heddleston, John M and Giang, William and Anthony, Neil and Chew, Teng-Leong and Gilbert-Ross, Melissa} } @article {67736, title = {Visualizing the Invisible: Advanced Optical Microscopy as a Tool to Measure Biomechanical Forces.}, journal = {Frontiers in Cell and Developmental Biology}, volume = {9}, year = {2021}, month = {2021 Sep 06}, pages = {706126}, abstract = {

The importance of mechanical force in biology is evident across diverse length scales, ranging from tissue morphogenesis during embryo development to mechanotransduction across single adhesion proteins at the cell surface. Consequently, many force measurement techniques rely on optical microscopy to measure forces being applied by cells on their environment, to visualize specimen deformations due to external forces, or even to directly apply a physical perturbation to the sample via photoablation or optogenetic tools. Recent developments in advanced microscopy offer improved approaches to enhance spatiotemporal resolution, imaging depth, and sample viability. These advances can be coupled with already existing force measurement methods to improve sensitivity, duration and speed, amongst other parameters. However, gaining access to advanced microscopy instrumentation and the expertise necessary to extract meaningful insights from these techniques is an unavoidable hurdle. In this Live Cell Imaging special issue Review, we survey common microscopy-based force measurement techniques and examine how they can be bolstered by emerging microscopy methods. We further explore challenges related to the accompanying data analysis in biomechanical studies and discuss the various resources available to tackle the global issue of technology dissemination, an important avenue for biologists to gain access to pre-commercial instruments that can be leveraged for biomechanical studies.

}, issn = {2296-634X}, doi = {10.3389/fcell.2021.706126}, author = {Hobson, Chad M and Aaron, Jesse S and Heddleston, John M and Chew, Teng-Leong} } @article {65602, title = {FMNL2 regulates dynamics of fascin in filopodia.}, journal = {Journal of Cell Biology}, volume = {219}, year = {2020}, month = {2020 May 04}, abstract = {

Filopodia are peripheral F-actin-rich structures that enable cell sensing of the microenvironment. Fascin is an F-actin-bundling protein that plays a key role in stabilizing filopodia to support efficient adhesion and migration. Fascin is also highly up-regulated in human cancers, where it increases invasive cell behavior and correlates with poor patient prognosis. Previous studies have shown that fascin phosphorylation can regulate F-actin bundling, and that this modification can contribute to subcellular fascin localization and function. However, the factors that regulate fascin dynamics within filopodia remain poorly understood. In the current study, we used advanced live-cell imaging techniques and a fascin biosensor to demonstrate that fascin phosphorylation, localization, and binding to F-actin are highly dynamic and dependent on local cytoskeletal architecture in cells in both 2D and 3D environments. Fascin dynamics within filopodia are under the control of formins, and in particular FMNL2, that binds directly to dephosphorylated fascin. Our data provide new insight into control of fascin dynamics at the nanoscale and into the mechanisms governing rapid cytoskeletal adaptation to environmental changes. This filopodia-driven exploration stage may represent an essential regulatory step in the transition from static to migrating cancer cells.

}, issn = {1540-8140}, doi = {10.1083/jcb.201906111}, author = {Pfisterer, Karin and Levitt, James and Lawson, Campbell D and Marsh, Richard J and Heddleston, John M and Wait, Eric and Ameer-Beg, Simon Morris and Cox, Susan and Parsons, Maddy} } @article {65601, title = {Frontline science: dynamic cellular and subcellular features of migrating leukocytes revealed by in vivo lattice lightsheet microscopy.}, journal = {Journal of Leukocyte Biology}, year = {2020}, month = {2020 Apr 23}, abstract = {

Neutrophil and macrophage (Mϕ) migration underpin the inflammatory response. However, the fast velocity, multidirectional instantaneous movement, and plastic, ever-changing shape of phagocytes confound high-resolution intravital imaging. Lattice lightsheet microscopy (LLSM) captures highly dynamic cell morphology at exceptional spatiotemporal resolution. We demonstrate the first extensive application of LLSM to leukocytes in vivo, utilizing optically transparent zebrafish, leukocyte-specific reporter lines that highlighted subcellular structure, and a wounding assay for leukocyte migration. LLSM revealed details of migrating leukocyte morphology, and permitted intricate, volumetric interrogation of highly dynamic activities within their native physiological setting. Very thin, recurrent uropod extensions must now be considered a characteristic feature of migrating neutrophils. LLSM resolved trailing uropod extensions, demonstrating their surprising length, and permitting quantitative assessment of cytoskeletal contributions to their evanescent form. Imaging leukocytes in blood vessel microenvironments at LLSM{\textquoteright}s spatiotemporal resolution displayed blood-flow-induced neutrophil dynamics and demonstrated unexpected leukocyte-endothelial interactions such as leukocyte-induced endothelial deformation against the intravascular pressure. LLSM of phagocytosis and cell death provided subcellular insights and uncovered novel behaviors. Collectively, we provide high-resolution LLSM examples of leukocyte structures (filopodia lamellipodia, uropod extensions, vesicles), and activities (interstitial and intravascular migration, leukocyte rolling, phagocytosis, cell death, and cytoplasmic ballooning). Application of LLSM to intravital leukocyte imaging sets the stage for transformative studies into the cellular and subcellular complexities of phagocyte biology.

}, issn = {1938-3673}, doi = {10.1002/JLB.3HI0120-589R}, author = {Manley, Harriet R and Potter, David L and Heddleston, John M and Chew, Teng-Leong and Keightley, M Cristina and Lieschke, Graham J} } @article {64784, title = {An actin-based protrusion originating from a podosome-enriched region initiates macrophage fusion.}, journal = {Molecular Biology of the Cell}, volume = {30}, year = {2019}, month = {2019 Aug 1}, pages = {2254-67}, abstract = {

Macrophage fusion resulting in the formation of multinucleated giant cells occurs in a variety of chronic inflammatory diseases, yet the mechanism responsible for initiating macrophage fusion is unknown. Here, we used live cell imaging to show that actin-based protrusions at the leading edge initiate macrophage fusion. Phase contrast video microscopy demonstrated that in the majority of events, short protrusions (3 {\textpm} 1 μm) between two closely apposed cells initiated fusion, but occasionally we observed long protrusions (16 {\textpm} 7 μm). Using macrophages isolated from LifeAct mice and imaging with lattice light sheet microscopy, we further found that fusion-competent actin-based protrusions formed at sites enriched in podosomes. Inducing fusion in mixed populations of GFP- and mRFP-LifeAct macrophages showed rapid spatial overlap between GFP and RFP signal at the site of fusion. Cytochalasin B strongly reduced fusion and when rare fusion events occurred, protrusions were not observed. Fusion of macrophages deficient in Wiskott-Aldrich syndrome protein and Cdc42, key molecules involved in the formation of actin-based protrusions and podosomes, was also impaired both in vitro and in vivo. Finally, inhibiting the activity of the Arp2/3 complex decreased fusion and podosome formation. Together these data indicate that an actin-based protrusion formed at the leading edge macrophage fusion.

}, doi = {10.1101/538314}, author = {Faust, James J and Balabiyev, Arnat and Heddleston, John M and Podolnikova, Nataly P and Baluch, Debra Page and Chew, Teng-Leong and Ugarova, Tatiana P} } @article {49582, title = {Characterization of neurite dystrophy after trauma by high speed structured illumination microscopy and lattice light sheet microscopy.}, journal = {Journal of Neuroscience Methods}, volume = {312}, year = {2019}, month = {2019 Jan 15}, pages = {154-61}, abstract = {

BACKGROUND: Unbiased screening studies have repeatedly identified actin-related proteins as one of the families of proteins most influenced by neurotrauma. Nevertheless, the status quo model of cytoskeletal reorganization after neurotrauma excludes actin and incorporates only changes in microtubules and intermediate filaments. Actin is excluded in part because it is difficult to image with conventional techniques. However, recent innovations in fluorescent microscopy provide an opportunity to image the actin cytoskeleton at super-resolution resolution in living cells. This study applied these innovations to an in vitro model of neurotrauma.

NEW METHOD: New methods are introduced for traumatizing neurons before imaging them with high speed structured illumination microscopy or lattice light sheet microscopy. Also, methods for analyzing structured illumination microscopy images to quantify post-traumatic neurite dystrophy are presented.

RESULTS: Human induced pluripotent stem cell-derived neurons exhibited actin organization typical of immature neurons. Neurite dystrophy increased after trauma but was not influenced by jasplakinolide treatment. The F-actin content of dystrophies varied greatly from one dystrophy to another.

COMPARISON WITH EXISTING METHODS: In contrast to fixation dependent methods, these methods capture the evolution of the actin cytoskeleton over time in a living cell. In contrast to prior methods based on counting dystrophies, this quantification scheme parameterizes the severity of a given dystrophy as it evolves from a local swelling to an almost-perfect spheroid that threatens to transect the neurite.

CONCLUSIONS: These methods can be used to investigate genetic factors and therapeutic interventions that modulate the course of neurite dystrophy after trauma.

}, issn = {1872-678X}, doi = {10.1016/j.jneumeth.2018.12.005}, author = {Phillips, Jack K and Sherman, Sydney A and Cotton, Kristen Y and Heddleston, John M and Taylor, Aaron B and Finan, John D} } @article {65175, title = {Histone H3K27 acetylation precedes active transcription during zebrafish zygotic genome activation as revealed by live-cell analysis.}, journal = {Development}, volume = {146}, year = {2019}, month = {2019 Sep 30}, abstract = {

Histone post-translational modifications are key gene expression regulators, but their rapid dynamics during development remain difficult to capture. We applied a Fab-based live endogenous modification labeling technique to monitor the changes in histone modification levels during zygotic genome activation (ZGA) in living zebrafish embryos. Among various histone modifications, H3 Lys27 acetylation (H3K27ac) exhibited most drastic changes, accumulating in two nuclear foci in the 64- to 1k-cell-stage embryos. The elongating form of RNA polymerase II, which is phosphorylated at Ser2 in heptad repeats within the C-terminal domain (RNAP2 Ser2ph), and miR-430 transcripts were also concentrated in foci closely associated with H3K27ac. When treated with α-amanitin to inhibit transcription or JQ-1 to inhibit binding of acetyl-reader proteins, H3K27ac foci still appeared but RNAP2 Ser2ph and miR-430 morpholino were not concentrated in foci, suggesting that H3K27ac precedes active transcription during ZGA. We anticipate that the method presented here could be applied to a variety of developmental processes in any model and non-model organisms.

}, issn = {1477-9129}, doi = {10.1242/dev.179127}, author = {Sato, Yuko and Hilbert, Lennart and Oda, Haruka and Wan, Yinan and Heddleston, John M and Chew, Teng-Leong and Zaburdaev, Vasily and Keller, Philipp and Lionnet, Timoth{\'e}e and Vastenhouw, Nadine and Kimura, Hiroshi} } @article {64851, title = {Interfacial actin protrusions mechanically enhance killing by cytotoxic T cells.}, journal = {Science Immunology}, volume = {4}, year = {2019}, month = {2019 Mar 22}, abstract = {

Cytotoxic T lymphocytes (CTLs) kill by forming immunological synapses with target cells and secreting toxic proteases and the pore-forming protein perforin into the intercellular space. Immunological synapses are highly dynamic structures that boost perforin activity by applying mechanical force against the target cell. Here, we used high-resolution imaging and microfabrication to investigate how CTLs exert synaptic forces and coordinate their mechanical output with perforin secretion. Using micropatterned stimulatory substrates that enable synapse growth in three dimensions, we found that perforin release occurs at the base of actin-rich protrusions that extend from central and intermediate locations within the synapse. These protrusions, which depended on the cytoskeletal regulator WASP and the Arp2/3 actin nucleation complex, were required for synaptic force exertion and efficient killing. They also mediated physical deformation of the target cell surface during CTL-target cell interactions. Our results reveal the mechanical basis of cellular cytotoxicity and highlight the functional importance of dynamic, three-dimensional architecture in immune cell-cell interfaces.

}, issn = {2470-9468}, doi = {10.1126/sciimmunol.aav5445}, author = {Tamzalit, Fella and Wang, Mitchell S and Jin, Weiyang and Tello-Lafoz, Maria and Boyko, Vitaly and Heddleston, John M and Black, Charles T and Kam, Lance C and Huse, Morgan} } @article {64853, title = {Membrane-cytoskeletal crosstalk mediated by myosin-I regulates adhesion turnover during phagocytosis.}, journal = {Nature Communications}, volume = {10}, year = {2019}, month = {2019 03 19}, pages = {1249}, abstract = {

Phagocytosis of invading pathogens or cellular debris requires a dramatic change in cell shape driven by actin polymerization. For antibody-covered targets, phagocytosis is thought to proceed through the sequential engagement of Fc-receptors on the phagocyte with antibodies on the target surface, leading to the extension and closure of the phagocytic cup around the target. We find that two actin-dependent molecular motors, class 1 myosins myosin 1e and myosin 1f, are specifically localized to Fc-receptor adhesions and required for efficient phagocytosis of antibody-opsonized targets. Using primary macrophages lacking both myosin 1e and myosin 1f, we find that without the actin-membrane linkage mediated by these myosins, the organization of individual adhesions is compromised, leading to excessive actin polymerization, slower adhesion turnover, and deficient phagocytic internalization. This work identifies a role for class 1 myosins in coordinated adhesion turnover during phagocytosis and supports a mechanism involving membrane-cytoskeletal crosstalk for phagocytic cup closure.

}, issn = {2041-1723}, doi = {10.1038/s41467-019-09104-1}, author = {Barger, Sarah R and Reilly, Nicholas S and Shutova, Maria S and Li, Qingsen and Maiuri, Paolo and Heddleston, John M and Mooseker, Mark S and Flavell, Richard A and Svitkina, Tatyana and Oakes, Patrick W and Krendel, Mira and Gauthier, Nils C} } @article {49056, title = {BAK/BAX macropores facilitate mitochondrial herniation and mtDNA efflux during apoptosis.}, journal = {Science (New York, N.Y.)}, volume = {359}, year = {2018}, month = {2018 02 23}, abstract = {

Mitochondrial apoptosis is mediated by BAK and BAX, two proteins that induce mitochondrial outer membrane permeabilization, leading to cytochrome c release and activation of apoptotic caspases. In the absence of active caspases, mitochondrial DNA (mtDNA) triggers the innate immune cGAS/STING pathway, causing dying cells to secrete type I interferon. How cGAS gains access to mtDNA remains unclear. We used live-cell lattice light-sheet microscopy to examine the mitochondrial network in mouse embryonic fibroblasts. We found that after BAK/BAX activation and cytochrome c loss, the mitochondrial network broke down and large BAK/BAX pores appeared in the outer membrane. These BAK/BAX macropores allowed the inner mitochondrial membrane to herniate into the cytosol, carrying with it mitochondrial matrix components, including the mitochondrial genome. Apoptotic caspases did not prevent herniation but dismantled the dying cell to suppress mtDNA-induced innate immune signaling.

}, issn = {1095-9203}, doi = {10.1126/science.aao6047}, author = {McArthur, Kate and Whitehead, Lachlan W and Heddleston, John M and Li, Lucy and Padman, Benjamin S and Oorschot, Viola and Geoghegan, Niall D and Chappaz, Stephane and Davidson, Sophia and San Chin, Hui and Lane, Rachael M and Dramicanin, Marija and Saunders, Tahnee L and Sugiana, Canny and Lessene, Romina and Osellame, Laura D and Chew, Teng-Leong and Dewson, Grant and Lazarou, Michael and Ramm, Georg and Lessene, Guillaume and Ryan, Michael T and Rogers, Kelly L and van Delft, Mark F and Kile, Benjamin T} } @article {49382, title = {Macropinosome formation by tent pole ruffling in macrophages.}, journal = {The Journal of Cell Biology}, volume = {217}, year = {2018}, month = {2018 Aug 27}, pages = {3873-85}, abstract = {

Pathogen-mediated activation of macrophages arms innate immune responses that include enhanced surface ruffling and macropinocytosis for environmental sampling and receptor internalization and signaling. Activation of macrophages with bacterial lipopolysaccharide (LPS) generates prominent dorsal ruffles, which are precursors for macropinosomes. Very rapid, high-resolution imaging of live macrophages with lattice light sheet microscopy (LLSM) reveals new features and actions of dorsal ruffles, which redefine the process of macropinosome formation and closure. We offer a new model in which ruffles are erected and supported by F-actin tent poles that cross over and twist to constrict the forming macropinosomes. This process allows for formation of large macropinosomes induced by LPS. We further describe the enrichment of active Rab13 on tent pole ruffles and show that CRISPR deletion of Rab13 results in aberrant tent pole ruffles and blocks the formation of large LPS-induced macropinosomes. Based on the exquisite temporal and spatial resolution of LLSM, we can redefine the ruffling and macropinosome processes that underpin innate immune responses.

}, issn = {1540-8140}, doi = {10.1083/jcb.201804137}, author = {Condon, Nicholas D and Heddleston, John M and Chew, Teng-Leong and Luo, Lin and McPherson, Peter S and Ioannou, Maria S and Hodgson, Louis and Stow, Jennifer L and Wall, Adam A} } @article {48607, title = {Cytoskeletal actin dynamics shape a ramifying actin network underpinning immunological synapse formation.}, journal = {Science Advances}, volume = {3}, year = {2017}, month = {2017 Jun 21}, pages = {e1603032}, abstract = {

T cell activation and especially trafficking of T cell receptor microclusters during immunological synapse formation are widely thought to rely on cytoskeletal remodeling. However, important details on the involvement of actin in the latter transport processes are missing. Using a suite of advanced optical microscopes to analyze resting and activated T cells, we show that, following contact formation with activating surfaces, these cells sequentially rearrange their cortical actin across the entire cell, creating a previously unreported ramifying actin network above the immunological synapse. This network shows all the characteristics of an inward-growing transportation network and its dynamics correlating with T cell receptor rearrangements. This actin reorganization is accompanied by an increase in the nanoscale actin meshwork size and the dynamic adjustment of the turnover times and filament lengths of two differently sized filamentous actin populations, wherein formin-mediated long actin filaments support a very flat and stiff contact at the immunological synapse interface. The initiation of immunological synapse formation, as highlighted by calcium release, requires markedly little contact with activating surfaces and no cytoskeletal rearrangements. Our work suggests that incipient signaling in T cells initiates global cytoskeletal rearrangements across the whole cell, including a stiffening process for possibly mechanically supporting contact formation at the immunological synapse interface as well as a central ramified transportation network apparently directed at the consolidation of the contact and the delivery of effector functions.

}, doi = {10.1126/sciadv.1603032}, author = {Fritzsche, Marco and Fernandes, Ricardo A and Chang, Veronica T and Colin-York, Huw and Clausen, Mathias P and Felce, James H and Galiani, Silvia and Erlenk{\"a}mper, Christoph and Santos, Ana M and Heddleston, John M and Pedroza-Pacheco, Isabela and Waithe, Dominic and de la Serna, Jorge Bernadino and Lagerholm, B Christoffer and Liu, Tsung-Li and Chew, Teng-Leong and Betzig, Eric and Davis, Simon J and Eggeling, Christian} } @article {48936, title = {A moving source of matrix components is essential for de novo basement membrane formation.}, journal = {Current Biology : CB}, volume = {27}, year = {2017}, month = {2017 Nov 02}, pages = {3526-34}, abstract = {

The basement membrane (BM) is a thin layer of extracellular matrix (ECM) beneath nearly all epithelial cell types that is critical for cellular and tissue function. It is composed of numerous components conserved among all bilaterians [1]; however, it is unknown how all of these components are generated and subsequently constructed to form a fully mature BM in the living animal. Although BM formation is thought to simply involve a process of self-assembly [2], this concept suffers from a number of logistical issues when considering its construction in\ vivo. First, incorporation of BM components appears to be hierarchical [3-5], yet it is unclear whether their production during embryogenesis must also be regulated in a temporal fashion. Second, many BM proteins are produced not only by the cells residing on the BM but also by surrounding cell types [6-9], and it is unclear how large, possibly insoluble protein complexes [10] are delivered into the matrix. Here we exploit our ability to live image and genetically dissect de novo BM formation during Drosophila development. This reveals that there is a temporal hierarchy of BM protein production that is essential for proper component incorporation. Furthermore, we show that BM components require secretion by migrating macrophages (hemocytes) during their developmental dispersal, which is critical for embryogenesis. Indeed, hemocyte migration is essential to deliver a subset of ECM components evenly throughout the embryo. This reveals that de novo BM construction requires a combination of both production and distribution logistics allowing for the timely delivery of core components.

}, issn = {1879-0445}, doi = {10.1016/j.cub.2017.10.001}, author = {Matsubayashi, Yutaka and Louani, Adam and Dragu, Anca and S{\'a}nchez-S{\'a}nchez, Besaiz J and Serna-Morales, Eduardo and Yolland, Lawrence and Gyoergy, Attila and Vizcay, Gema and Fleck, Roland A and Heddleston, John M and Chew, Teng-Leong and Siekhaus, Daria E and Stramer, Brian M} } @article {48866, title = {Nanoscale visualization of biomineral formation in coral proto-polyps.}, journal = {Current Biology : CB}, volume = {27}, year = {2017}, month = {2017 Oct 11}, pages = {3191-6}, abstract = {

Calcium carbonate platforms produced by reef-building stony corals over geologic time are pervasive features around the world [1]; however, the mechanism by which these organisms produce the mineral is poorly understood (see review by [2]). It is generally assumed that stony corals precipitate calcium carbonate extracellularly as aragonite in a calcifying medium between the calicoblastic ectoderm and pre-existing skeleton, separated from the overlying seawater [2]. The calicoblastic ectoderm produces extracellular matrix (ECM) proteins, secreted to the calcifying medium [3-6], which appear to provide the nucleation, alteration, elongation, and inhibition mechanisms of the biomineral [7] and remain occluded and preserved in the skeleton [8-10]. Here we show in cell cultures of the stony coral Stylophora pistillata that calcium is concentrated in intracellular pockets that are subsequently exported from the cell where a nucleation process leads to the formation of extracellular aragonite crystals. Analysis of the growing crystals by lattice light-sheet microscopy suggests that the crystals elongate from the cells{\textquoteright} surfaces outward.

}, issn = {1879-0445}, doi = {10.1016/j.cub.2017.09.012}, author = {Mass, Tali and Drake, Jeana L and Heddleston, John M and Falkowski, Paul G} } @article {48992, title = {Protein-templated gold nanoparticle synthesis: protein organization, controlled gold sequestration, and unexpected reaction products.}, journal = {Dalton Transactions (Cambridge, England : 2003)}, volume = {46}, year = {2017}, month = {2017 Dec 21}, pages = {16465-16473}, abstract = {

Emerging applications that exploit the properties of nanoparticles for biotechnology require that the nanoparticles be biocompatible or support biological recognition. These types of particles can be produced through syntheses that involve biologically relevant molecules (proteins or natural extracts, for example). Many of the protocols that rely on these molecules are performed without a clear understanding of the mechanism by which the materials are produced. We have investigated a previously described reaction in which gold nanoparticles are produced from the reaction of chloroauric acid and proteins in solution. We find that modifications to the starting conditions can alter the product from the expected solution-suspended colloids to a product where colloids are formed within a solid, fibrous protein structure. We have interrogated this synthesis, exploiting the change in products to better understand this reaction. We have evaluated the kinetics and products for 7 different proteins over a range of concentrations and temperatures. The key factor that controls the synthetic outcome (colloid or fiber) is the concentration of the protein relative to the gold concentration. We find that the observed fibrous structures are more likely to form at low protein concentrations and when hydrophilic proteins are used. An analysis of the reaction kinetics shows that AuNP formation occurs faster at lower protein (fiber-forming) concentrations than at higher protein (colloid-forming) concentrations. These results contradict traditional expectations for reaction kinetics and protein-fiber formation and are instructive of the manner in which proteins template gold nanoparticle production.

}, issn = {1477-9234}, doi = {10.1039/c7dt03275g}, author = {Hart, Cassidy and Abuladel, Nouf and Bee, Madeleine and Kreider, Megan C and CVitan, Alexander C and Esson, Moira M and Farag, Andrew and Ibeh, Trisha and Kalivas, Eleni N and Larco, Daniel-Mario and Walker Long, Andrew and Lymperopoulos, Loukas and Mendel, Zachary and Miles, Nancy and Zareba, Carly M and Schwabacher, James C and Slucher, Helen and Vinals, Javier and Heddleston, John M and Li, Wenyue and Fox, Douglas M and Hartings, Matthew R} } @article {48206, title = {Developmentally programmed germ cell remodelling by endodermal cell cannibalism.}, journal = {Nature Cell Biology}, volume = {18}, year = {2016}, month = {2016 Dec}, pages = {1302-10}, abstract = {

Primordial germ cells (PGCs) in many species associate intimately with endodermal cells, but the significance of such interactions is largely unexplored. Here, we show that Caenorhabditis elegans PGCs form lobes that are removed and digested by endodermal cells, dramatically altering PGC size and mitochondrial content. We demonstrate that endodermal cells do not scavenge lobes PGCs shed, but rather, actively remove lobes from the cell body. CED-10 (Rac)-induced actin, DYN-1 (dynamin) and LST-4 (SNX9) transiently surround lobe necks and are required within endodermal cells for lobe scission, suggesting that scission occurs through a mechanism resembling vesicle endocytosis. These findings reveal an unexpected role for endoderm in altering the contents of embryonic PGCs, and define a form of developmentally programmed cell remodelling involving intercellular cannibalism. Active roles for engulfing cells have been proposed in several neuronal remodelling events, suggesting that intercellular cannibalism may be a more widespread method used to shape cells than previously thought.

}, issn = {1476-4679}, doi = {10.1038/ncb3439}, author = {Abdu, Yusuff and Maniscalco, Chelsea and Heddleston, John M and Chew, Teng-Leong and Nance, Jeremy} } @article {47937, title = {Light sheet microscopes: Novel imaging toolbox for visualizing life{\textquoteright}s processes.}, journal = {The International Journal of Biochemistry \& Cell Biology}, year = {2016}, month = {2016 Oct 7}, abstract = {

Capturing dynamic processes in live samples is a nontrivial task in biological imaging. Although fluorescence provides high specificity and contrast compared to other light microscopy techniques, the photophysical principles of this method can have a harmful effect on the sample. Current advances in light sheet microscopy have created a novel imaging toolbox that allows for rapid acquisition of high-resolution fluorescent images with minimal perturbation of the processes of interest. Each unique design has its own advantages and limitations. In this review, we describe several cutting edge light sheet microscopes and their optimal applications.

}, issn = {1878-5875}, doi = {10.1016/j.biocel.2016.10.002}, author = {Heddleston, John M and Chew, Teng-Leong} } @article {46819, title = {High-resolution live imaging reveals axon-glia interactions during peripheral nerve injury and repair in zebrafish.}, journal = {Disease Models \& Mechanisms}, volume = {8}, year = {2015}, month = {2015 Jun 1}, pages = {553-64}, abstract = {

Neural damage is a devastating outcome of physical trauma. The glia are one of the main effectors of neuronal repair in the nervous system, but the dynamic interactions between peripheral neurons and Schwann cells during injury and regeneration remain incompletely characterized. Here, we combine laser microsurgery, genetic analysis, high-resolution intravital imaging and lattice light-sheet microscopy to study the interaction between Schwann cells and sensory neurons in a zebrafish model of neurotrauma. We found that chronic denervation by neuronal ablation leads to Schwann-cell death, whereas acute denervation by axonal severing does not affect the overall complexity and architecture of the glia. Neuronal-circuit regeneration begins when Schwann cells extend bridging processes to close the injury gap. Regenerating axons grow faster and directionally after the physiological clearing of distal debris by the Schwann cells. This might facilitate circuit repair by ensuring that axons are guided through unoccupied spaces within bands of B{\"u}ngner towards their original peripheral target. Accordingly, in the absence of Schwann cells, regenerating axons are misrouted, impairing the re-innervation of sensory organs. Our results indicate that regenerating axons use haptotaxis as a directional cue during the reconstitution of a neural circuit. These findings have implications for therapies aimed at neurorepair, which will benefit from preserving the architecture of the peripheral glia during periods of denervation.

}, issn = {1754-8411}, doi = {10.1242/dmm.018184}, author = {Xiao, Yan and Faucherre, Ad{\`e}le and Pola-Morell, Laura and Heddleston, John M and Liu, Tsung-Li and Chew, Teng-Leong and Sato, Fuminori and Sehara-Fujisawa, Atsuko and Kawakami, Koichi and L{\'o}pez-Schier, Hern{\'a}n} }