A bacterial aphrodisiac, tissue origami, amyloid proteins protecting dormant eggs, and exactly how the Zika virus damages cells—these discoveries are among the ten top research abstracts selected by ASCB’s Public Information Committee (PIC) for its Honor list of “novel & newsworthy” science being presented December 3-7 at ASCB 2016 in San Francisco.
The scientist-members of ASCB’s PIC plowed through the 1263 research abstracts submitted for panel presentation in San Francisco, winnowing the pile down in two complete rounds to these 10 Honor picks. For ASCB 2016 attendees, times, dates, and location for these presentations are listed. News media should contact the authors directly.
Amyloid-like Self-Assembly of a Cellular Compartment
- Boke1, M. Ruer2, M. Wuhr1,3, M. Coughlin1, R. Lemaitre2, S.P. Gygi3, S. Alberti2, D.N. Drechsel2, A.A. Hyman2, T.J. Mitchison1;
1Department of Systems Biology, Harvard Medical School, Boston, MA, 2Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany, 3Department of Cell Biology, Harvard Medical School, Boston, MA
Microsymposium Talk E82, Microsymposium 10: Spatial Organization of the Cell, Monday, December 5, 12:28-12:35 pm
A key characteristic of female germ cells, oocytes, is that their complement of mitochondria and RNA is kept intact for decades before fertilization. However, we have little idea how these are protected for such long periods of time. One prominent feature of dormant oocytes is the Balbiani body, which is conserved from fish to humans. These enigmatic structures, which can be thought of as a non-membranous compartment, or perhaps super-organelle, are prominent in the cytoplasm of early oocytes, and are packed with mitochondria, ER, Golgi and RNA. Very little is known about their organization and structure.
We have studied the organization of the Balbiani body in Xenopus, the large size of which makes it amenable to biochemistry. We show by quantitative mass spectrometry that the most enriched protein in the Balbiani body of Xenopus is Xvelo, a homolog of the germ line protein Bucky ball in zebrafish. Xvelo has a prion-like domain in its N terminus, which is sufficient and necessary to target to the Balbiani body. We show that Xvelo forms a mitochondria-embedding, amyloid-like matrix pervading the entire volume of the Balbiani body. Moreover, recombinant Xvelo forms micron-sized networks in vitro that can cluster mitochondria on its own in a cell free system in a prion-like domain dependent manner, thus reconstituting aspects of a Balbiani body in vitro.
We propose that the Balbiani body forms by amyloid-like aggregation of Xvelo. Because the prion- like domain of Xvelo is conserved in different germ plasm-related proteins in other species, Balbiani body formation by amyloid-like assembly could be a conserved feature in evolution for maintaining the immortal character of germ cells.
TRAPPC11 mutations lead to a diverse set of disorders with muscular dystrophy as a common feature
- Sacher1,2, M.P. Milev2, K. Prematilake2, A. Larson3, S. Moore4, K. Köhler5, A. Hübner5, C. Jimenez-Mallabrera6;
1Anatomy and Cell Biology, McGill University, Montreal, QC, 2Biology, Concordia University, Montreal, QC, 3Pediatrics, Children’s Hospital of Colorado, Aurora, CO, 4Pathology, University of Iowa, Iowa City, IA, 5Children, Dresden University of Technology, Dresden, Germany, 6Neuromuscular Unit, Hospital Sant Joan de Déu, Barcelona, Spain
Poster P412, ER and Golgi Transport, Sunday, December 4, 1:30-3:00 pm, Board B972
Transport Protein Particle (TRAPP) is composed of numerous proteins that are distributed in at least two related complexes in humans. These complexes function along the endomembrane system and mediate transport between various compartments including endoplasmic reticulum-to-Golgi, intra- Golgi and endocytosis, as well as in autophagy. The TRAPPC11 gene encodes a subunit of TRAPP and has been implicated in transport in the early secretory pathway as well as in autophagy. More recently, this subunit was also implicated in lipid-linked oligosaccharide (LLO) synthesis, suggesting that it may have a function outside of the complex that affects protein glycosylation. Three different mutations in TRAPPC11 have been previously reported and these patients displayed a variety of phenotypes including cataracts, steatosis, ataxia, intellectual disability, congenital muscular dystrophy, myopathy and limb girdle muscular dystrophy 2S (LGMD2S). Here we present several patients harboring three additional homozygous and compound heterozygous TRAPPC11 mutations. One patient has a Triple A-like disorder with alacrima, achalasia, scoliosis, dystrophic changes and cerebral atrophy. A second patient has congenital muscular dystrophy with documented dystroglycanopathy including brain atrophy and retinal disease as well as hepatic steatosis. The third patient displays cognitive impairment, muscular dystrophy and microcephaly. Collectively, TRAPPC11 mutations are linked to a diverse set of clinical phenotypes in the spectrum of muscular dystrophy with many extra-muscular manifestations. TRAPPC11 should therefore be considered in the diagnostic evaluation of patients with neuromuscular disorders and brain involvement of unknown genetic origin particularly when linked to dystroglycanopathy and Triple A-like disorders.
Tissue origami: Directed folding of tissues by programmed cell contractility networks
A.J. Hughes1, M.C. Coyle1, J. Zhang1, Z.J. Gartner1; 1Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA
Minsymposium Talk M110, Minisymposium 10: Connecting Cells to Tissues, Monday, December 5, 6:35-6:50 pm
A major barrier to the engineering of tissues to study development is the difficulty in controlling tissue structure across a wide range of length scales. Further, proper function of synthetic biological tissues is hampered by mass transport limitations, and the lack of extracellular matrix (ECM) physicochemical anisotropies that guide appropriate cell behaviors in space and time.
In biology, tissues are typically built over a range of length scales via self-organized morphogenesis processes in which extracellular matrix remodeling occurs at the same time as cell division, migration, and fate specification. For example, the specification of endoderm cell fate at crypts in the gut is governed by cell-cell signaling networks that are influenced by the folding state of tissue layers.
We take a biomimetic approach to building tissues by engineering the spatial organization of sheets of ECM gels via contractile networks of cells. Such contractile networks are co-patterned with “passenger” cell types that can be guided into prescribed 3D topologies that instruct cell self- organization processes. Thus, the folding trajectory and form is combined with control over the type and placement of different cell types on the sheet, enabling control over 3D tissue architecture in concert with cellular interfaces.
We build arrays of contractile tissues at the upper and lower surface of 250 micron-thick matrigel- collagen ECM-mimetic gels using DNA-patterned assembly of cells. The type and location of cells on each surface of the gel prescribe the final folded geometry of the ECM.
Regular arrays of contractile mammary epithelial cells placed on one side of circular ECM gels yield cap-shaped objects with maximum curvatures that increase over time. The curvature at a given timepoint increases monotonically as the spacing between tissues on the sheet is decreased (and more tissues are added), scales linearly with cell contractility, and is partially blocked by blebbistatin. We found that anisotropic grids of fibroblast tissues produce consistent anisotropic folds along controlled axes, allowing us to fabricate a range of 3D topographies and predict them with a biophysical tensegrity model. For example, we constructed the Miura-ori fold, which resembles folds of the gut epithelium in development.
We report engineering control over the 3D shape of biomimetic ECM scaffolds by leveraging tissues as mechanical actuators. Our ability to design specific fold geometries in concert with cell compositions and spatial arrangement in ECM sheets could enable studies of the necessary conditions for topological transitions in animal development.
Membrane fission by protein crowding
W.T. Snead1, C.C. Hayden1, A.K. Gadok1, P. Rangamani2, J.C. Stachowiak1;
1Biomedical Engineering, The University of Texas at Austin, Austin, TX, 2Mechanical and Aerospace Engineering, University of California, San Diego, La Jolla, CA
Minisymposium Talk M237, Minisymposium 23: Membrane Traffic Control by Cytoskeleton and Molecular Machines, Wednesday, December 7, 10:05-10:20 am
Compartmentalization of biological processes into discrete, membrane-bound volumes is essential for cellular life. The process of membrane fission drives the formation of new compartments from initially continuous membrane surfaces. Proteins with specific structural features and assembly properties, including helical scaffolds and hydrophobic insertions, are traditionally viewed as the primary mediators of fission in the cell. In contrast, here we report a previously unknown mechanism of membrane fission, which is independent of protein shape and assembly – protein crowding. Specifically, when proteins strongly adhere to the membrane surface at high coverage, in-plane collisions among diffusing proteins generate substantial surface pressure. If this pressure is not balanced by an equivalent pressure on the opposite membrane surface, the resulting gradient in surface pressure dramatically increases membrane curvature, leading to spontaneous membrane fission. By correlating FRET-based measurements of the membrane coverage by adhered proteins with multiple, independent measurements of membrane fission including membrane shedding, fluorescence-based quantification of vesicle size, and electron microscopy, we show that both fission efficiency and the size of fission products are correlated with the coverage of protein on the membrane. Specifically, using Epsin1, a clathrin adaptor previously thought to promote fission through insertion of a wedge-like hydrophobic helix into the membrane, we show that membrane coverage is the main determinant of fission efficiency and vesicle size, regardless of the hydrophobicity of the inserting helix. Importantly, we show that increasing the size of the adhered protein leads to increased fission efficiency, in line with a crowding mechanism driving fission. Our data contrast starkly with the established view that hydrophobic insertions drive membrane fission directly. Instead, we show that hydrophobic insertions are more important for strongly anchoring proteins to the membrane surface, to create highly crowded coverages of protein that favor fission. Further, our results provide a potential explanation for how fission may have been achieved in early cells, before the evolution of complex fission machines like dynamin. In particular, our findings demonstrate that any protein, regardless of its structure or assembly properties, can in principle drive fission, suggesting that the basic energetic criteria for membrane fission may be met by a much greater variety of proteins than previously thought.
Zika infection disrupts centriole biogenesis.
A.T. Kodani1,2, J.F. Reiter2, K. Knopp2, J. DeRisi2;
1Genetics and Genomics, Boston Children’s Hospital, Boston, MA, 2Biochemistry and Biophysics, UCSF, San Francisco, CA
Microsymposium Talk E149, Microsymposium 17: Cell Division in Development and Disease, Tuesday, December 6, 2:21-2:28 pm
Zika virus (ZIKV) is an arbovirus transmitted to humans by mosquito bites and sexual transmission. Outbreaks of the virus in Brazil and Central America have been linked to a rise in congenital neurodevelopmental conditions, including microcephaly and Guillain-Barre syndrome. Recent studies have revealed that ZIKV can infect human neural stem cells growing as brain organoids and abrogate neurogenesis in mice; however, the cellular mechanism by which brain development is disrupted remains unknown. Here we demonstrate that the Brazilian and Costa Rican ZIKV strains disrupt centriole biogenesis, a phenotype commonly associated with the autosomal recessive disorder, primary microcephaly (MCPH). Cells infected with ZIKV produce supernumerary Centrin foci that over-accumulate the MCPH protein CEP63 and CCDC14, critical regulators of centriole duplication. We demonstrate that CEP63 and CCDC14 interact with the Zika protease/helicase, NS3. Moreover, expression of the active form of NS3 leads to the formation of overabundant Centrin foci that accumulate CEP63 and CCDC14. These results suggest that the neurodevelopmental defects in ZIKV infected patients arise as a result of abrogated centriole biogenesis.
PhotoGate Microscopy for Tracking Single Molecules in Crowded Environments
- Belyy1,2, S. Shih3, J. Bandaria3, Y. Huang1, R. Lawrence4, R. Zoncu4, A. Yildiz1,3,4;
1Biophysics, University of California, Berkeley, Berkeley, CA, 2Biophysics, University of California, San Francisco, San Francisco, CA, 3Department of Physics, University of California, Berkeley, Berkeley, CA, 4Department of Molecular Cell Biology, University of California, Berkeley, Berkeley, CA
Microsymposium Talk M157, Microsymposium 10: Spatial Organization of the Cell, Monday, December 5, 1:17-1:24 pm
Tracking single molecules inside cells reveals the dynamics of biological processes, including receptor trafficking, signaling and cargo transport. However, individual molecules often cannot be resolved inside cells due to their high density. We developed the PhotoGate technique that controls the number of fluorescent particles in a region of interest by repeatedly photobleaching its boundary with a steerable focused laser beam. PhotoGate bypasses the requirement of photoactivation to track single particles at surface densities two orders of magnitude greater than the single-molecule detection limit. Using this method, we observed ligand-induced dimerization of a receptor tyrosine kinase at the cell surface and directly measured binding and dissociation of signaling molecules from early endosomes in a dense cytoplasm with single molecule resolution. We additionally developed a numerical simulation of PhotoGate that models diffusion and photobleaching from first principles and allows users to optimize experimental parameters prior to performing the experiment.
Genetic and infectious causes of microcephaly caused by NDE1 mutations and Zika virus
D.J. Doobin1, A. Rosenfeld2, A. Carabalona1, V. Racaniello2, R.B. Vallee1;
1Pathology and Cell Biology, Columbia University Medical Center, New York, NY, 2Microbiology and Immunology, Columbia University Medical Center, New York, NY
Minisymposium Talk M157, Minisymposium 15: Cell Polarity & Morphogenesis , Tuesday, December 6, 5:50-6:05 pm
Microcephaly is a brain developmental malformation characterized by an abnormally small neocortex and head size. Recent work from our lab has elucidated the cellular mechanisms responsible for a severe form of microcephaly caused by human mutations in NDE1, a protein involved in cytoplasmic dynein regulation. Using in utero electroporation of NDE1 short-hairpin RNA in embryonic rat brain, we have observed three independent points of severe cell cycle arrest in proliferating neural progenitors. These occur during regulation of primary cilia at the G1-to-S transition, during G2- dependent apical interkinetic nuclear migration (INM), and at the G2-to-M transition (Doobin et al., Nature Commun., 2016, in press). Overexpression of NDEL1, a NDE1 paralogue, could functionally compensate for NDE1 loss, except at the G2-to-M transition, suggesting a novel role for NDE1 in mitotic entry in this system. To gain insight into the mechanisms controlling NDE1 behavior during the neural progenitor cell cycle we have begun to examine the effects of NDE1 phosphorylation by Cdk1. We find that expression of a NDE1 double phosphomutant is sufficient to block apical INM, whereas a single site phosphomutant causes the G2-M arrest. These results reveal that the severity of NDE1-associated microcephaly results not from defects in mitosis, but rather the inability of neural progenitors to ever reach this stage. By applying this understanding of microcephaly with a similar methodology to organotypic mouse brain slices, we have begun to investigate the mechanism of Zika virus-induced microcephaly. Using diverse isolates of Zika virus we observe differences in viral pathology in mouse embryonic brain slice preparations, though nearly all strains can replicate in this system. We observe a severe decrease in mitotic events in the mouse brain progenitor cells, and evidence for subsequent defects in postmitotic neuronal migration and viability as well. By studying both genetic and infectious causes of microcephaly, we hope to obtain a more complete understanding of the cell biology of this disorder, and that of other malformations of cortical development. Supp. by HD40182 and NINDS F30NS095577.
A bacterially-produced aphrodisiac regulates choanoflagellate mating
- Woznica1, J.P. Gerdt2, J. Clardy2, N. King1;
1Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, 2BCMP , Harvard Medical School, Boston, MA
Minisymposium Talk M162, Microsymposium 162, Dark Matters in Signaling & Differentiation, Tuesday, December 6, 4:35-4:50 pm
The earliest animals evolved in oceans teeming with bacteria, and bacterial symbionts continue to profoundly shape the biology of their animal hosts. Choanoflagellates, the closest living relatives of animals, provide a simple and evolutionarily relevant model to help uncover fundamental mechanisms by which bacteria influence animal biology. We recently discovered that the bacterium, Vibrio fischeri, induces mating in the choanoflagellate, S. rosetta. Within minutes of exposure to Vibrio bacteria, S. rosetta haploid cells form mating-swarms through cell aggregation. Pairs of swarming cells then mate, undergoing cell and nuclear fusion, followed by meiotic recombination. We have determined that the mating induction factor produced by Vibrio is a secreted chondroitin sulfate (CS) lyase that is sufficient to induce choanoflagellate mating at nanomolar concentrations. Interestingly, similar extracellular glycosaminoglycan lyases play essential roles in sperm-egg binding and fertilization in diverse animals, from sea stars to humans. This discovery that a bacterial cue regulates choanoflagellate mating is the first example of bacteria driving mating in eukaryotes, and raises the possibility that environmental bacteria or bacterial symbionts regulate mating in animals as well.
Schwann cells activated by cancer cells induce cancer cell invasion
S… Deborde1, T.A. Omelchenko2, A. Lyubchik1, Y. Zhou1, S. He1, W. McNamara1, N. Chernichenko1, S. Lee1, C. Chen1, R. Bakst1, E. Vakiani1, S. He1, A. Hall2, R.J. Wong1;
1Surgery, Memorial Sloan Kettering Cancer Center, New YOrk, NY, 2Cell Biology, Memorial Sloan Kettering Cancer Center, New York, NY
Poster P324, Tumor Microenvironment 1, Sunday, December 4, 1:30-3:00 pm. B550
The nerve microenvironment facilitates cancer progression. Innervation promotes cancer growth and cancer cells derived from several cancer types, including pancreatic cancer, can extend along nerves through the process of perineural invasion, which is associated with poor prognosis. Our goal is to understand how nerves contribute to cancer progression. Here, we show that the main nerve cell type, the Schwann cells, promote cancer invasion through direct cancer cell contact and that Schwann cells are directly activated by cancer cells. A sub-population of Schwann cells expressing glial fibrillary acidic protein (GFAP) was associated with cancer cells in murine and human histological specimen of perineural invasion. GFAP expression was directly induced by cancer cells in in vitro experiments. Real time imaging revealed that murine Schwann cells from dorsal root ganglion in culture with cancer cells direct cancer cells to migrate toward nerves and promote invasion in a contact-dependent manner; and that human cultured Schwann cells establish contact with cancer cells and stimulate their dispersion. Upon contact, Schwann cells induce the formation of cancer cell protrusions in their direction and intercalate between the cancer cells, leading to cancer cell dispersion. The molecular mechanism of this process involves Schwann cell neural cell adhesion molecule 1 (NCAM1). The reported Schwann cell behavior reflects normal Schwann cell programs that are typically activated in nerve repair but are instead induced by cancer cells and exploited by cancer cells to promote perineural invasion and cancer progression.
Interplay of the Microbiome and Innate Immune System in Tissue Regeneration
C.P. Arnold1, S. Merryman1, A. Harris-Arnold1, C. Seidel1, S. McKinney1, A. Sánchez Alvarado1; 1Stowers Institute of Medical Research, Kansas City, MO
Poster P2420, Host-Pathogen/Host-Commensal Interactions 2, Tuesday, December 6, 1:30 -3:00 pm, Board B1424
The interrelationship between endogenous microbiota, the immune system, and tissue regeneration is an area of intense research due to its potential therapeutic applications. We investigated this relationship in the planaria Schmidtea mediterranea, a model organism capable of regenerating any and all of its adult tissues. Microbiome characterization revealed a high Bacteroidetes to Proteobacteria ratio in healthy planarians, a composition analogous to that of the human intestinal tract but not conserved in other invertebrate or even some vertebrate model organisms. Perturbations eliciting an expansion of Proteobacteria in planaria coincided with ectopic lesions and tissue degeneration. Culture of these bacteria yielded a strain of Pseudomonas capable of inducing progressive tissue degeneration. Remarkably, a subset of planaria chronically infected with Pseudomonas were capable of resolving their wounds to regenerate degenerated tissues including large portions of the central nervous system. RNAi screening uncovered a T AK1 innate immune signaling module underlying compromised tissue homeostasis and regeneration during infection. Given the complex role of inflammation in either the hindrance or support of reparative wound healing and regeneration, this invertebrate model provides a basis for dissecting the duality of evolutionarily conserved inflammatory signaling in complex, multi-organ adult tissue regeneration. Furthermore, it provides a novel means of inquiry into the role of the microbiome in host tissue regeneration.