@article {68512, title = {Oligodendrocyte precursor cells ingest axons in the mouse neocortex.}, journal = {Proceedings of the National Academies of Science of the U.S.A.}, volume = {119}, year = {2022}, month = {2022 Nov 29}, pages = {e2202580119}, abstract = {

Neurons in the developing brain undergo extensive structural refinement as nascent circuits adopt their mature form. This physical transformation of neurons is facilitated by the engulfment and degradation of axonal branches and synapses by surrounding glial cells, including microglia and astrocytes. However, the small size of phagocytic organelles and the complex, highly ramified morphology of glia have made it difficult to define the contribution of these and other glial cell types to this crucial process. Here, we used large-scale, serial section transmission\ electron microscopy (TEM) with computational volume segmentation to reconstruct the complete 3D morphologies of distinct glial types in the mouse visual cortex, providing unprecedented resolution of their morphology and composition. Unexpectedly, we discovered that the fine processes of oligodendrocyte precursor cells (OPCs), a population of abundant, highly dynamic glial progenitors, frequently surrounded small branches of axons. Numerous phagosomes and phagolysosomes (PLs) containing fragments of axons and vesicular structures were present inside their processes, suggesting that OPCs engage in axon pruning. Single-nucleus RNA sequencing from the developing mouse cortex revealed that OPCs express key phagocytic genes at this stage, as well as neuronal transcripts, consistent with active axon engulfment. Although microglia are thought to be responsible for the majority of synaptic pruning and structural refinement, PLs were ten times more abundant in OPCs than in microglia at this stage, and these structures were markedly less abundant in newly generated oligodendrocytes, suggesting that OPCs contribute substantially to the refinement of neuronal circuits during cortical development.

}, keywords = {Animals, Axons, Mice, Neocortex, Neurons, Oligodendrocyte Precursor Cells, Oligodendroglia}, issn = {1091-6490}, doi = {10.1073/pnas.2202580119}, author = {Buchanan, JoAnn and Elabbady, Leila and Collman, Forrest and Jorstad, Nikolas L and Bakken, Trygve E and Ott, Carolyn and Glatzer, Jenna and Bleckert, Adam A and Bodor, Agnes L and Brittain, Derrick and Bumbarger, Daniel J and Mahalingam, Gayathri and Seshamani, Sharmishtaa and Schneider-Mizell, Casey and Takeno, Marc M and Torres, Russel and Yin, Wenjing and Hodge, Rebecca D and Castro, Manuel and Dorkenwald, Sven and Ih, Dodam and Jordan, Chris S and Kemnitz, Nico and Lee, Kisuk and Lu, Ran and Macrina, Thomas and Mu, Shang and Popovych, Sergiy and Silversmith, William M and Tartavull, Ignacio and Turner, Nicholas L and Wilson, Alyssa M and Wong, William and Wu, Jingpeng and Zlateski, Aleksandar and Zung, Jonathan and Lippincott-Schwartz, Jennifer and Lein, Ed S and Seung, H Sebastian and Bergles, Dwight E and Reid, R Clay and da Costa, Nuno Ma{\c c}arico} } @article {49457, title = {Noncanonical autophagy at ER exit sites regulates procollagen turnover.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {115}, year = {2018}, month = {2018 Oct 04}, pages = {E10099-108}, abstract = {

Type I collagen is the main component of bone matrix and other connective tissues. Rerouting of its procollagen precursor to a degradative pathway is crucial for osteoblast survival in pathologies involving excessive intracellular buildup of procollagen that is improperly folded and/or trafficked. What cellular mechanisms underlie this rerouting remains unclear. To study these mechanisms, we employed live-cell imaging and correlative light and electron microscopy (CLEM) to examine procollagen trafficking both in wild-type mouse osteoblasts and osteoblasts expressing a bone pathology-causing mutant procollagen. We found that although most procollagen molecules successfully trafficked through the secretory pathway in these cells, a subpopulation did not. The latter molecules appeared in numerous dispersed puncta colocalizing with COPII subunits, autophagy markers and ubiquitin machinery, with more puncta seen in mutant procollagen-expressing cells. Blocking endoplasmic reticulum exit site (ERES) formation suppressed the number of these puncta, suggesting they formed after procollagen entry into ERESs. The punctate structures containing procollagen, COPII, and autophagic markers did not move toward the Golgi but instead were relatively immobile. They appeared to be quickly engulfed by nearby lysosomes through a bafilomycin-insensitive pathway. CLEM and fluorescence recovery after photobleaching experiments suggested engulfment occurred through a noncanonical form of autophagy resembling microautophagy of ERESs. Overall, our findings reveal that a subset of procollagen molecules is directed toward lysosomal degradation through an autophagic pathway originating at ERESs, providing a mechanism to remove excess procollagen from cells.

}, issn = {1091-6490}, doi = {10.1073/pnas.1814552115}, author = {Omari, Shakib and Makareeva, Elena and Roberts-Pilgrim, Anna and Mirigian, Lynn and Jarnik, Michal and Ott, Carolyn and Lippincott-Schwartz, Jennifer and Leikin, Sergey} } @article {49058, title = {VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis.}, journal = {Scientific Reports}, volume = {8}, year = {2018}, month = {2018 Feb 20}, pages = {3353}, abstract = {

The hexameric AAA ATPase VPS4 facilitates ESCRT III filament disassembly on diverse intracellular membranes. ESCRT III components and VPS4 have been localized to the ciliary transition zone and spindle poles and reported to affect centrosome duplication and spindle pole stability. How the canonical ESCRT pathway could mediate these events is unclear. We studied the association of VPS4 with centrosomes and found that GFP-VPS4 was a dynamic component of both mother and daughter centrioles. A mutant, VPS4, which can{\textquoteright}t hydrolyze ATP, was less dynamic and accumulated at centrosomes. Centrosome localization of the VPS4mutant, caused reduced γ-tubulin levels at centrosomes and consequently decreased microtubule growth and altered centrosome positioning. In addition, preventing VPS4 ATP hydrolysis nearly eliminated centriolar satellites and paused ciliogensis after formation of the ciliary vesicle. Zebrafish embryos injected with GFP-VPS4mRNA were less viable, exhibited developmental defects and had fewer cilia in Kupffer{\textquoteright}s vesicle. Surprisingly, ESCRT III proteins seldom localized to centrosomes and their depletion did not lead to these phenotypes. Our data support an ESCRT III-independent function for VPS4 at the centrosome and reveal that this evolutionary conserved AAA ATPase influences diverse centrosome functions and, as a result, global cellular architecture and development.

}, issn = {2045-2322}, doi = {10.1038/s41598-018-21491-x}, author = {Ott, Carolyn and Nachmias, Dikla and Adar, Shai and Jarnik, Michal and Sherman, Shachar and Birnbaum, Ramon Y and Lippincott-Schwartz, Jennifer and Elia, Natalie} } @article {48116, title = {Sonic Hedgehog Pathway activation increases mitochondrial abundance and activity in hippocampal neurons.}, journal = {Molecular Biology of the Cell}, year = {2016}, month = {2016 Dec 08}, abstract = {

Mitochondria are essential organelles whose biogenesis, structure, and function are regulated by many signaling pathways. In this study we present evidence that, in hippocampal neurons, activation of the Sonic hedgehog (Shh) signaling pathway impacts multiple aspects of mitochondria. Mitochondrial mass was increased significantly in neurons treated with Shh. Using biochemical and fluorescence imaging analyses, we show that Shh signaling activity reduces mitochondrial fission and promotes mitochondrial elongation, at least in part, via suppression of the mitochondrial fission protein dynamin-like GTPase Drp1. Mitochondria from Shh-treated neurons were more electron-dense as revealed by electron microscopy, and had higher membrane potential and respiratory activity. We further show that Shh protects neurons against a variety of stresses, including the mitochondrial poison rotenone, amyloid β-peptide, hydrogen peroxide, and high levels of glutamate. Collectively, our data suggest a link between Shh pathway activity and the physiological properties of mitochondria in hippocampal neurons.

}, issn = {1939-4586}, doi = {10.1091/mbc.E16-07-0553}, author = {Yao, Pamela J and Manor, Uri and Petralia, Ronald S and Brose, Rebecca D and Wu, Ryan T Y and Ott, Carolyn and Wang, Ya-Xian and Charnoff, Ari and Lippincott-Schwartz, Jennifer and Mattson, Mark P} } @article {47467, title = {Early steps in primary cilium assembly require EHD1/EHD3-dependent ciliary vesicle formation.}, journal = {Nature Cell Biology}, volume = {17}, year = {2015}, month = {2015 Feb 16}, pages = {228-40}, abstract = {

Membrane association with mother centriole (M-centriole) distal appendages is critical for ciliogenesis initiation. How the Rab GTPase Rab11{\textendash}​Rab8\ cascade functions in early ciliary membrane assembly is unknown. Here, we show that the membrane shaping proteins ​EHD1\ and ​EHD3, in association with the Rab11{\textendash}​Rab8\ cascade, function in early ciliogenesis. ​EHD1\ and ​EHD3\ localize to preciliary membranes and the ciliary pocket. EHD-dependent membrane tubulation is essential for ciliary vesicle formation from smaller distal appendage vesicles (DAVs). Importantly, this step functions in M-centriole to basal body transformation and recruitment of transition zone proteins and ​IFT20. ​SNAP29, a SNARE membrane fusion regulator and ​EHD1-binding protein, is also required for DAV-mediated ciliary vesicle assembly. Interestingly, only after ciliary vesicle assembly is ​Rab8\ activated for ciliary growth. Our studies uncover molecular mechanisms informing a previously uncharacterized ciliogenesis step, whereby ​EHD1\ and ​EHD3\ reorganize the M-centriole and associated DAVs before coordinated ciliary membrane and axoneme growth.

}, issn = {1476-4679}, doi = {10.1038/ncb3109}, author = {Lu, Quanlong and Insinna, Christine and Ott, Carolyn and Stauffer, Jimmy and Pintado, Petra A and Rahajeng, Juliati and Baxa, Ulrich and Walia, Vijay and Cuenca, Adrian and Hwang, Yoo-Seok and Daar, Ira O and Lopes, Susana and Lippincott-Schwartz, Jennifer and Jackson, Peter K and Caplan, Steve and Westlake, Christopher J} } @article {47629, title = {A contractile and counterbalancing adhesion system controls the 3D shape of crawling cells.}, journal = {The Journal of cell biology}, volume = {205}, year = {2014}, month = {2014 Apr 14}, pages = {83-96}, abstract = {

How adherent and contractile systems coordinate to promote cell shape changes is unclear. Here, we define a counterbalanced adhesion/contraction model for cell shape control. Live-cell microscopy data showed a crucial role for a contractile meshwork at the top of the cell, which is composed of actin arcs and myosin IIA filaments. The contractile actin meshwork is organized like muscle sarcomeres, with repeating myosin II filaments separated by the actin bundling protein α-actinin, and is mechanically coupled to noncontractile dorsal actin fibers that run from top to bottom in the cell. When the meshwork contracts, it pulls the dorsal fibers away from the substrate. This pulling force is counterbalanced by the dorsal fibers{\textquoteright} attachment to focal adhesions, causing the fibers to bend downward and flattening the cell. This model is likely to be relevant for understanding how cells configure themselves to complex surfaces, protrude into tight spaces, and generate three-dimensional forces on the growth substrate under both healthy and diseased conditions.

}, keywords = {Actin Cytoskeleton, Actinin, Actins, Animals, Cell Adhesion, Cell Line, Tumor, Cell Movement, Cell Shape, Cercopithecus aethiops, COS Cells, Focal Adhesions, Humans, Mechanotransduction, Cellular, Models, Biological, Nonmuscle Myosin Type IIA, Pressure, Time Factors, Transfection}, issn = {1540-8140}, doi = {10.1083/jcb.201311104}, author = {Burnette, Dylan T and Shao, Lin and Ott, Carolyn and Pasapera, Ana M and Fischer, Robert S and Baird, Michelle A and Der Loughian, Christelle and Delanoe-Ayari, Helene and Paszek, Matthew J and Davidson, Michael W and Betzig, Eric and Lippincott-Schwartz, Jennifer} } @article {45153, title = {A contractile and counterbalancing adhesion system controls the 3D shape of crawling cells.}, journal = {Journal of Cell Biology}, volume = {205}, year = {2014}, month = {2014 Apr 14}, pages = {83-96}, abstract = {

How adherent and contractile systems coordinate to promote cell shape changes is unclear. Here, we define a counterbalanced adhesion/contraction model for cell shape control. Live-cell microscopy data showed a crucial role for a contractile meshwork at the top of the cell, which is composed of actin arcs and myosin IIA filaments. The contractile actin meshwork is organized like muscle sarcomeres, with repeating myosin II filaments separated by the actin bundling protein α-actinin, and is mechanically coupled to noncontractile dorsal actin fibers that run from top to bottom in the cell. When the meshwork contracts, it pulls the dorsal fibers away from the substrate. This pulling force is counterbalanced by the dorsal fibers{\textquoteright} attachment to focal adhesions, causing the fibers to bend downward and flattening the cell. This model is likely to be relevant for understanding how cells configure themselves to complex surfaces, protrude into tight spaces, and generate three-dimensional forces on the growth substrate under both healthy and diseased conditions.

}, keywords = {Actin Cytoskeleton, Actinin, Actins, Animals, Cell Adhesion, Cell Line, Tumor, Cell Movement, Cell Shape, Cercopithecus aethiops, COS Cells, Focal Adhesions, Humans, Mechanotransduction, Cellular, Models, Biological, Nonmuscle Myosin Type IIA, Pressure, Time Factors, Transfection}, issn = {1540-8140}, doi = {10.1083/jcb.201311104}, author = {Burnette, Dylan T and Shao, Lin and Ott, Carolyn and Pasapera, Ana M and Fischer, Robert S and Baird, Michelle A and Der Loughian, Christelle and Delanoe-Ayari, Helene and Paszek, Matthew J and Davidson, Michael W and Betzig, Eric and Lippincott-Schwartz, Jennifer} } @article {47634, title = {Incisive imaging and computation for cellular mysteries: lessons from abscission.}, journal = {Cell}, volume = {155}, year = {2013}, month = {2013 Dec 5}, pages = {1220-31}, abstract = {

The final cleavage event that terminates cell division, abscission of the small, dense intercellular bridge, has been particularly challenging to resolve. Here, we describe imaging innovations that helped answer long-standing questions about the mechanism of abscission. We further explain how computational modeling of high-resolution data was employed to test hypotheses and generate additional insights. We present the model that emerges from application of these complimentary approaches. Similar experimental strategies will undoubtedly reveal exciting details about other underresolved cellular structures.

}, keywords = {Animals, Cell Physiological Processes, Cytokinesis, Humans, Models, Biological, Plant Cells}, issn = {1097-4172}, doi = {10.1016/j.cell.2013.11.011}, author = {Elia, Natalie and Ott, Carolyn and Lippincott-Schwartz, Jennifer} } @article {47645, title = {Visualization of live primary cilia dynamics using fluorescence microscopy.}, journal = {Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]}, volume = {Chapter 4}, year = {2012}, month = {2012 Dec}, pages = {Unit 4.26}, abstract = {

Methods useful for exploring the formation and functions of primary cilia in living cells are described here. First, multiple protocols for visualizing solitary cilia that extend away from the cell body are described. Primary cilia collect, synthesize, and transmit information about the extracellular space into the cell body to promote critical cellular responses. Problems with cilia formation or function can lead to dramatic changes in cell physiology. These methods can be used to assess cilia formation and length, the location of the cilium relative to other cellular structures, and localization of specific proteins to the cilium. The subsequent protocols describe how to quantify movement of fluorescent molecules within the cilium using kymographs, photobleaching, and photoconversion. The microtubules that form the structural scaffold of the cilium are also critical avenues for kinesin and dynein-mediated movement of proteins within the cilium. Assessing intraflagellar dynamics can provide insight into mechanisms of ciliary-mediated signal perception and transmission.

}, keywords = {Animals, Bodily Secretions, Cilia, Dyneins, Epithelial Cells, Flagella, Humans, Kinesin, Kymography, Microscopy, Fluorescence, Microtubules, Photobleaching, Primary Cell Culture, Signal Transduction}, issn = {1934-2616}, doi = {10.1002/0471143030.cb0426s57}, author = {Ott, Carolyn and Lippincott-Schwartz, Jennifer} } @article {47654, title = {Visualization of live primary cilia dynamics using fluorescence microscopy.}, journal = {Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]}, volume = {Chapter 4}, year = {2012}, month = {2012 Dec}, pages = {Unit 4.26}, abstract = {

Methods useful for exploring the formation and functions of primary cilia in living cells are described here. First, multiple protocols for visualizing solitary cilia that extend away from the cell body are described. Primary cilia collect, synthesize, and transmit information about the extracellular space into the cell body to promote critical cellular responses. Problems with cilia formation or function can lead to dramatic changes in cell physiology. These methods can be used to assess cilia formation and length, the location of the cilium relative to other cellular structures, and localization of specific proteins to the cilium. The subsequent protocols describe how to quantify movement of fluorescent molecules within the cilium using kymographs, photobleaching, and photoconversion. The microtubules that form the structural scaffold of the cilium are also critical avenues for kinesin and dynein-mediated movement of proteins within the cilium. Assessing intraflagellar dynamics can provide insight into mechanisms of ciliary-mediated signal perception and transmission.

}, keywords = {Animals, Bodily Secretions, Cilia, Dyneins, Epithelial Cells, Flagella, Humans, Kinesin, Kymography, Microscopy, Fluorescence, Microtubules, Photobleaching, Primary Cell Culture, Signal Transduction}, issn = {1934-2616}, doi = {10.1002/0471143030.cb0426s57}, author = {Ott, Carolyn and Lippincott-Schwartz, Jennifer} }