Assaying stem cell mechanobiology on microfabricated elastomeric substrates with geometrically modulated rigidity (11-Jan)
Michael T Yang, Jianping Fu, Yang-Kao Wang, Ravi A Desai1 & Christopher S Chen
Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, USA
We describe the use of a microfabricated cell culture substrate, consisting of a uniform array of closely spaced, vertical, elastomeric microposts, to study the effects of substrate rigidity on cell function. Elastomeric micropost substrates are micromolded from silicon masters comprised of microposts of different heights to yield substrates of different rigidities. The tips of the elastomeric microposts are functionalized with extracellular matrix through microcontact printing to promote cell adhesion. These substrates, therefore, present the same topographical cues to adherent cells while varying substrate rigidity only through manipulation of micropost height. this protocol describes how to fabricate the silicon micropost array masters (~2 weeks to complete) and elastomeric substrates (3 d), as well as how to perform cell culture experiments (1-14 d), immunofluorescence imaging (2 d), traction force analysis (2 d) and stem cell differentiation assays (1 d) on these substrates in order to examine the effect of substrate rigidity on stem cell morphology, traction force generation, focal adhesion organization and differentiation.
Ultrastructural analysis and identification of membrane proteins in the free-living amoeba Difflugia corona (6-Dec)
MARCELO SILVA-BRIANO 1, SANDRA LUZ MARTÍNEZ-HERNÁNDEZ 1, ARACELI ADABACHE-ORTÍZ 1, JAVIER VENTURAJUÁREZ 2, EVA SALINAS 3, J. LUIS QUINTANAR 4
1 Dept. of Biology, 2 Dept. Morphology, 3 Dept. de Microbiology and 4 Dept. of Physiology and Pharmacology. Centro de Ciencias Básicas, Universidad Autónoma de Aguascalientes. Av. Universidad 940 C.P. 20100. Aguascalientes Ags. México.
Syntaxin-1 and 25-kDa Synaptosome-associated Protein (SNAP-25) are present in the plasma membrane of several different secretory cell types and are involved in the exocytosis process. In this work, the free-living amoeba Difflugia corona was studied in relation to ultrastructure, structural membrane proteins, and proteins such as Syntaxin-1 and SNAP-25. Our results obtained by scanning electron microscopy in the amoeba without its theca, showed many membrane projections and several pore-like structures. Using immunocytochemistry, we found structural proteins Syntaxin-1 and SNAP-25.
Key Words: amoeba, membrane proteins, pore-like, theca, ultrastructure
Coupled myosin VI motors facilitate unidirectional movement on an F-actin network (9-Oct)
Sivaraj Sivaramakrishnan and James A. Spudich
Department of Biochemistry, Stanford University, Stanford, CA 94305 USA
Unconventional myosins interact with the dense cortical actin network during processes such as membrane trafficking, cell migration, and mechanotransduction. Our understanding of unconventional myosin function is derived largely from assays that examine the interaction of a single myosin with a single actin filament. In this study, we have developed a model system to study the interaction between multiple tethered unconventional myosins and a model F-actin cortex, namely the lamellipodium of a migrating fish epidermal keratocyte. Using myosin VI, which moves toward the pointed end of actin filaments, we directly determine the polarity of the extracted keratocyte lamellipodium from the cell periphery to the cell nucleus. We use a combination of experimentation and simulation to demonstrate that multiple myosin VI molecules can coordinate to efficiently transport vesiclesize cargo over 10 µm of the dense interlaced actin network. Furthermore, several molecules of monomeric myosin VI, which are nonprocessive in single molecule assays, can coordinate to transport cargo with similar speeds as dimers.
Key Words: TMR, tetramethylrhodamine
Identification of Novel Serine-Threonine Protein Phosphatases in Trypanosoma cruzi: a Potential Role in Control of Cytokinesis and Morphology (1999)
GEORGE A. ORR 1, CRAIG WERNER 2, JUN XU 3, MARCIA BENNETT 1, LOUIS M. WEISS 2,3,4, PETER TAKVORKAN 5, HERBERT B. TANOWITZ 2,3,4, AND MURRAY WITTNER 2,3,4
Departments of Molecular Pharmacology 1, Pathology 3, and Medicine 4, Albert Einstein College of Medicine, Bronx, New York 10461; Division of Infectious Diseases, Albert Einstein College of Medicine and Montefiore Medical Center, Bronx, New York 10467 2; and Department of Biological Sciences, Rutgers University, Newark, New Jersey 07102 5
We cloned two novel Trypanosoma cruzi proteins by using degenerate oligonucleotide primers prepared against conserved domains in mammalian serine/threonine protein phosphatases 1, 2A, and 2B. The isolated genes encoded proteins of 323 and 330 amino acids, respectively, that were more homologous to the catalytic subunit of human protein phosphatase 1 than to those of human protein phosphatase 2A or 2B. The proteins encoded by these genes have been tentatively designated TcPP1a and TcPP1 (3. Northern blot analysis revealed the presence of a major 2.3-kb mRNA transcript hybridizing to each gene in both the epimastigote and metacyclic trypomastigote developmental stages. Southern blot analysis suggests that each protein phospha- tase 1 gene is present as a single copy in the T. cruzi genome. The complete coding region for TcPP1 (3 was expressed in Escherichia coli by using a vector, pTACTAC, with the trp-lac hybrid promoter. The recombinant protein from the TcPP1 (3 construct displayed phosphatase activity toward phosphorylase a, and this activity was preferentially inhibited by calyculin A (50% inhibitory concentration [IC50], -2 nM) over okadaic acid (IC50, -100 nM). Calyculin A, but not okadaic acid, had profound effects on the in vitro replication and morphology of T. cruzi epimastigotes. Low concentrations of calyculin A (1 to 10 nM) caused growth arrest. Electron microscopic studies of the calyculin A-treated epimastigotes revealed that the organisms underwent duplication of organelles, including the flagellum, kinetoplast, and nucleus, but were incapable of completing cell division. At concentrations higher than 10 nM, or upon prolonged incubation at lower concentrations, the epimastigotes lost their characteristic elongated spindle shape and had a more rounded morphology. Okadaic acid at concentrations up to 1 11M did not result in growth arrest or morphological alterations to T. cruzi epimastigotes. Calyculin A, but not okadaic acid, was also a potent inhibitor of the dephosphorylation of 32P-labeled phosphorylase a by T. cruzi epimastigotes and metacyclic trypomastigote extracts. These inhibitor studies suggest that in T. cruzi, type 1 protein phosphatases are important for the completion of cell division and for the maintenance of cell shape.
Nanometric self-assembling peptide layers maintain adult hepatocyte phenotype in sandwich cultures (2010)
Jonathan Wu 1, Núria Marí-Buyé 2,3, Teresa Fernández Muiños 2, Salvador Borrós 3, Pietro Favia 4, Carlos E Semino 1,2,5
1 Center for Biomedical Engineering, Massachusetts Institute of Technology, Boston, MA, USA.
2 Department of Bioengineering, Tissue Engineering Laboratory, IQS-Universidad Ramon Llull, Barcelona, Spain.
3 Grup d'Enginyeria de Materials, IQS-Universidad Ramon Llull, Barcelona, Spain.
4 Department of Chemistry, University of Bari, Italy.
5 Translational Centre for Regenerative Medicine (TRM-Leipzig), Universität Leipzig, Leipzig, Germany
Background: Isolated hepatocytes removed from their microenvironment soon lose their hepatospecific functions when cultured. Normally hepatocytes are commonly maintained under limited culture medium supply as well as scaffold thickness. Thus, the cells are forced into metabolic stress that degenerate liver specific functions. This study aims to improve hepatospecific activity by creating a platform based on classical collagen sandwich cultures.
Results: The modified sandwich cultures replace collagen with self-assembling peptide, RAD16-I, combined with functional peptide motifs such as the integrin-binding sequence RGD and the laminin receptor binding sequence YIG to create a cell-instructive scaffold. In this work, we show that a plasma-deposited coating can be used to obtain a peptide layer thickness in the nanometric range, which in combination with the incorporation of functional peptide motifs have a positive effect on the expression of adult hepatocyte markers including albumin, CYP3A2 and HNF4-alpha.
Conclusions: This study demonstrates the capacity of sandwich cultures with modified instructive self-assembling peptides to promote cell-matrix interaction and the importance of thinner scaffold layers to overcome mass transfer problems. We believe that this bioengineered platform improves the existing hepatocyte culture methods to be used for predictive toxicology and eventually for hepatic assist technologies and future artificial organs.
PKHD1L1 is a coat protein of hair-cell stereociliaand is required for normal hearing (2019)
Xudong Wu1,2,6, Maryna V. Ivanchenko1,6, Hoor Al Jandal1,3, Marcelo Cicconet4, Artur A. Indzhykulian1,5, David P. Corey1
1 Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA,
2 Decibel Therapeutics, 1325 Boylston Street,Boston, MA 02215, USA,
3 Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA
4 Image and Data Analysis Core, Harvard MedicalSchool, 43 Shattuck St, Boston, MA 02115, USA
5 Department of Otolaryngology, Harvard Medical School and Massachusetts Eye and Ear, 243 Charles St,Boston, MA 02114, USA
6 These authors contributed equally: Xudong Wu, Maryna V. Ivanchenko. Correspondence and requests for materials should beaddressed to A.A.I. (email:inartur@hms.harvard.edu) or to D.P.C. (email:dcorey@hms.harvard.edu)
The bundle of stereocilia on inner ear hair cells responds to subnanometer deflections pro-duced by sound or head movement. Stereocilia are interconnected by a variety of links andalso carry an electron-dense surface coat. The coat may contribute to stereocilia adhesion orprotect from stereocilia fusion, but its molecular identity remains unknown. From a databaseof hair-cell-enriched translated proteins, we identify Polycystic Kidney and Hepatic Disease 1-Like 1 (PKHD1L1), a large, mostly extracellular protein of 4249 amino acids with a singletransmembrane domain. Using serial immunogold scanning electron microscopy, we showthat PKHD1L1 is expressed at the tips of stereocilia, especially in the high-frequency regionsof the cochlea. PKHD1L1-deficient mice lack the surface coat at the upper but not lowerregions of stereocilia, and they develop progressive hearing loss. We conclude that PKHD1L1is a component of the surface coat and is required for normal hearing in mice.
Ocean acidification changes the structure of an Antarctic coastal protistan community (Jun 2017)
Alyce M. Hancock1,2,3, Andrew T. Davidson3,4, John McKinlay4, Andrew McMinn1,2,3, Kai G. Schulz5, and Rick L. van den Enden4
1Institute of Marine and Antarctic Studies, University of Tasmania, 20 Castray Esplanade, Battery Point TAS 7004, Australia
2Antarctic Gateway Partnership, 20 Castray Esplanade, Battery Point TAS 7004, Australia
3Antarctic Climate & Ecosystems Cooperative Research Centre, 20 Castray Esplanade, Battery Point TAS 7004, Australia
4Australian Antarctic Division, 203 Channel Hwy, Kingston TAS 7050, Australia
5Centre for Coastal Biogeochemistry, School of Environment, Science and Engineering, Southern Cross University, Lismore, NSW 2480, Australia
Antarctic near-shore waters are amongst the most sensitive in the world to ocean acidification. Microbes occupying these waters are critical drivers of ecosystem productivity, elemental cycling and ocean biogeochemistry, yet little is known about their sensitivity to ocean acidification. A six-level, dose-response experiment was conducted using 650 L incubation tanks (minicosms) adjusted to a gradient in fugacity of carbon dioxide (fCO2) from 343 to 1641 µatm. The six minicosms were filled with near-shore water from Prydz Bay, East Antarctica, and the protistan composition and abundance was determined by microscopy during 18 days of incubation. No CO2-related change in the protistan community composition was observed during the initial 8 day acclimation period under low light. Thereafter, the response of both autotrophic and heterotrophic protists to fCO2 was species-specific. The response of diatoms was mainly cell size related; microplanktonic diatoms (> 20 µm) increased in abundance with low to moderate fCO2 (343-634 µatm) but decreased at fCO2 ≥ 953 µatm. Similarly, the abundance of Phaeocystis antarctica increased with increasing fCO2 peaking at 634 µatm. Above this threshold the abundance of micro-sized diatoms and P. antarctica fell dramatically, and nanoplanktonic diatoms (≤ 20 µm) dominated, therefore culminating in a significant change in the protistan community composition. Comparisons of these results with previous experiments conducted at this site show that the fCO2 thresholds are similar, despite seasonal and interannual differences in the physical and biotic environment. This suggests that near-shore microbial communities are likely to change significantly near the end of this century if anthropogenic CO2 release continues unabated, with profound ramifications for near-shore Antarctic ecosystem food webs and biogeochemical cycling.
