Browsing by Subject "cell biology"

Now showing 1 - 16 of 16

Attractive membrane domains control lateral diffusion
(2008-05) Forstner, Martin B.; Martin, Douglas S.; Ruckerl, Florian; Kas, Josef A.; Selle, Carsten; Forstner, Martin B.; Martin, Douglas S.
Lipid membranes play a fundamental role in vital cellular functions such as signal transduction. Many of these processes rely on lateral diffusion within the membrane, generally a complex fluid containing ordered microdomains. However, little attention has been paid to the alterations in transport dynamics of a diffusing species caused by long-range interactions with membrane domains. In this paper, we address the effect of such interactions on diffusive transport by studying lateral diffusion in a phase-separated Langmuir phospholipid monolayer via single-particle tracking. We find that attractive dipole-dipole interactions between condensed phase domains and diffusing probe beads lead to transient confinement at the phase boundaries, causing a transition from two- to one-dimensional diffusion. Using Brownian dynamics simulations, the long-term diffusion constant for such a system is found to have a sensitive, Boltzmann-like, dependence on the interaction strength. In addition, this interaction strength is shown to be a strong function of the ratio of domain to particle size. As similar interactions are expected in biological membranes, the modulation of. diffusive transport dynamics by varying interaction strength and/or domain size may offer cells selective spatial and temporal control over signaling processes.
Functional Activation of ATM by the Prostate Cancer Suppressor NKX3.1
(2013-08) Cai, Bowen W.; Ju, J. H.; Lee, Jeong-Ho; Paull, Tanya T.; Gelmann, Edward P.; Lee, Jeong-Ho; Paull, Tanya T.
The prostate tumor suppressor NKX3.1 augments response to DNA damage and enhances survival after DNA damage. Within minutes of DNA damage, NKX3.1 undergoes phosphorylation at tyrosine 222, which is required for a functional interaction with ataxia telangiectasia mutated (ATM) kinase. NKX3.1 binds to the N-terminal region of ATM, accelerates ATM activation, and hastens the formation of gamma histone2AX. NKX3.1 enhances DNA-dependent ATM kinase activation by both the MRN complex and H2O2 in a DNA-damage-independent manner. ATM, bound to the NKX3.1 homeodomain, phosphorylates NKX3.1, leading to ubiquitination and degradation. Thus, NKX3.1 and ATM have a functional interaction leading to ATM activation and then NKX3.1 degradation in a tightly regulated DNA damage response specific to prostate epithelial cells. These findings demonstrate a mechanism for the tumor-suppressor properties of NKX3.1, demonstrate how NKX3.1 may enhance DNA integrity in prostate stem cells and may help to explain how cells differ in their sensitivity to DNA damage.
Generating lightly immortalizing T-Cells to study the host immune response and immunotherapies
(2023-04) Hoffman, Ashley S.; Umlauf, Benjamin J.
Immortalized T-cell lines are key tools for studying disease response, immune pathways, and novel treatments. However, many of these lines are transformed into undefined cancerous cells to maintain unlimited growth potential, limiting their utility as an accurate model of host T-cells. This creates a need for T-cell lines that mimic healthy behavior, especially for testing T-cell-mediated therapies (e.g., CAR T-cell and checkpoint inhibitor) and T-cell exhaustion, a phenomenon observed in patients undergoing intense chronic therapies, that can still be shared amongst the scientific community. To this end, we have developed a method of lightly immortalizing T-cells from human donor peripheral blood monocytes (PBMCs), without producing a cancerous phenotype, by only expressing 2 genes: human telomerase reverse transcriptase (hTERT) and cyclin-dependent kinase 4 (Cdk4). Cdk4 and hTERT are key genes in driving cell cycle and survival. Over expression of these genes should create a slowly dividing T-cell population that is still capable of responding to immune cues and signals. Lightly immortalized T-cells will serve as a key resource for studying T-cell-mediated therapies such as chimeric antigen receptor (CAR) T-cells and effects of chronic treatment, including T-cell exhaustion. Here, we present a robust method for lightly immortalizing T-cells from PBMCs. Our protocol successfully extends the lifespan of PBMCs and selects for effector and helper T-cell populations in two donor PBMCs. To verify successful transfection and selection of T-cells hTERT and Cdk4 we used western blotting and FACs. We also observed typical T-cell behavior post-immortalization and achieved a resting population of healthy, actively dividing T-cells with measuring expression of CD3. In conclusion, this method provides a straightforward, reproducible, and shareable tool for understanding T-cell response to treatment and studying T-cell exhaustion in multiple patients.
Global Transcriptional Profiling Reveals Distinct Functions of Thymic Stromal Subsets and Age-Related Changes During Thymic Involution
(2014-10) Ki, Sanghee; Park, Daechan; Selden, Hillary J.; Seita, Jun; Chung, Haewon; Kim, Jonghwan; Iyer, Vishwanath R.; Ehrlich, Lauren I. R.; Ki, Sanghee; Park, Daechan; Selden, Hillary J.; Chung, Haewon; Kim, Jonghwan; Iyer, Vishwanath R.; Ehrlich, Lauren I. R.
Age-associated thymic involution results in diminished T cell output and function in aged individuals. However, molecular mediators contributing to the decline in thymic function during early thymic involution remain largely unknown. Here, we present transcriptional profiling of purified thymic stromal subsets from mice 1, 3, and 6 months of age spanning early thymic involution. The data implicate unanticipated biological functions for a subset of thymic epithelial cells. The predominant transcriptional signature of early thymic involution is decreased expression of cell-cycle-associated genes and E2F3 transcriptional targets in thymic epithelial subsets. Also, expression of proinflammatory genes increases with age in thymic dendritic cells. Many genes previously implicated in late involution are already deregulated by 3-6 months of age. We provide these thymic stromal data sets, along with thymocyte data sets, in a readily searchable web-based platform, as a resource for investigations into thymocyte: stromal interactions and mechanisms of thymic involution.
Identification and characterization of novel ciliogenic machinery
(2017-05) Huizar, Ryan; Wallingford, John
Cilia are microtubule-based structures that project from almost every cell in the vertebrate body. In humans, there are two types of cilia, motile, which generate fluid flow across tissues of the ventricles, airway, and oviduct, as well as in propulsion in single cells, and primary, which are responsible for transducing many signaling pathways. Primary and motile cilia are dependent on a bidirectional trafficking process called intraflagellar transport (IFT) in order to bring material into the cilium, which governs their growth, maintenance, and signaling. IFT is mediated by two distinct protein complexes called IFT-A and IFT-B, which function in anterograde and retrograde transport, respectively. In motile cilia, an organization of multiple large protein complexes within the axoneme allow for wave-like motion to be produced. Instrumental to this motility are axonemal dynein arms, large motor protein complexes that slide along microtubule doublets in a coordinated manner to generate bending. Here, I describe two studies regarding ciliogenesis in multiciliated cells, a highly-specialized cell type decorated with dozens of motile cilia. First, I identify ANKRD55 as an IFT-B interactor. I demonstrate that this protein traffics through multiciliated cell axonemes and results in severe developmental defects in its absence. In addition, I describe early insights into the potential role this gene plays in cilia-related human disease. Together, these data suggest that ANKRD55 is a novel member of IFT-B. Second, I characterize the processes that underlie the cytoplasmic assembly of axonemal dynein arms, wherein various chaperones and cytoplasmic factors work in unison to fold and complex dynein arm subunits prior to ciliary transport. Using various imaging methods, I show that the factors responsible for dynein arm assembly localize to non-membrane bound cytoplasmic phase- separations in multiciliated cells, which we term DynAPs (Dynein Assembly Particles). I then demonstrate that machinery involved in phase separation of stress granules is required for formation of DynAPs and recruitment of dynein to axonemes.
Impaired Dendritic Expression and Plasticity Of H-Channels in the fmr1(-/Y) Mouse Model of Fragile X Syndrome
(2012-03) Brager, Darrin H.; Akhavan, Arvin R.; Johnston, Daniel; Brager, Darrin H.; Akhavan, Arvin R.; Johnston, Daniel
Despite extensive research into both synaptic and morphological changes, surprisingly little is known about dendritic function in fragile X syndrome (FXS). We found that the dendritic input resistance of CA1 neurons was significantly lower in fmr1(-/y) versus wild-type mice. Consistent with elevated dendritic I-h, voltage sag, rebound, and resonance frequency were significantly higher and temporal summation was lower in the dendrites of fmr1(-/y) mice. Dendritic expression of the h-channel subunit HCN1, but not HCN2, was higher in the CA1 region of fmr1(-/y) mice. Interestingly, whereas mGluR-mediated persistent decreases in Ih occurred in both wildtype and fmr1(-/y) mice, persistent increases in Ih that occurred after LTP induction in wild-type mice were absent in fmr1(-/y) mice. Thus, chronic upregulation of dendritic Ih in conjunction with impairment of homeostatic h-channel plasticity represents a dendritic channelopathy in this model of mental retardation and may provide a mechanism for the cognitive impairment associated with FXS.
Improved Ribosome-Footprint and mRNA Measurements Provide Insights into Dynamics and Regulation of Yeast Translation
(2016-02) Weinberg, David E.; Shah, Premal; Eichhorn, Steven W.; Hussmann, Jeffrey A.; Plotkin, Joshua B.; Bartel, David P.; Hussmann, Jeffrey A.
Ribosome-footprint profiling provides genome-wide snapshots of translation, but technical challenges can confound its analysis. Here, we use improved methods to obtain ribosome-footprint profiles and mRNA abundances that more faithfully reflect gene expression in Saccharomyces cerevisiae. Our results support proposals that both the beginning of coding regions and codons matching rare tRNAs are more slowly translated. They also indicate that emergent polypeptides with as few as three basic residues within a ten-residue window tend to slow translation. With the improved mRNA measurements, the variation attributable to translational control in exponentially growing yeast was less than previously reported, and most of this variation could be predicted with a simple model that considered mRNA abundance, upstream open reading frames, cap-proximal structure and nucleotide composition, and lengths of the coding and 50 UTRs. Collectively, our results provide a framework for executing and interpreting ribosome-profiling studies and reveal key features of translational control in yeast.
Intrinsically Disordered Segments affect Protein Half-Life in the Cell and During Evolution
(2014-09) van der Lee, Robin; Lang, Benjamin; Kruse, Kai; Gsponer, Jorg; de Groot, Natalia S.; Huynen, Martin A.; Matouschek, Andreas; Fuxreiter, Monika; Babu, M. Madan; Matouschek, Andreas
Precise control of protein turnover is essential for cellular homeostasis. The ubiquitin-proteasome system is well established as a major regulator of protein degradation, but an understanding of how inherent structural features influence the lifetimes of proteins is lacking. We report that yeast, mouse, and human proteins with terminal or internal intrinsically disordered segments have significantly shorter half-lives than proteins without these features. The lengths of the disordered segments that affect protein half-life are compatible with the structure of the proteasome. Divergence in terminal and internal disordered segments in yeast proteins originating from gene duplication leads to significantly altered half-life. Many paralogs that are affected by such changes participate in signaling, where altered protein half-life will directly impact cellular processes and function. Thus, natural variation in the length and position of disordered segments may affect protein half-life and could serve as an underappreciated source of genetic variation with important phenotypic consequences.
Ishemia-Reperfusion Enhances Gapdh Nitration In Aging Skeletal Muscle
(2011-10) Bailey, C. E.; Hammers, D. W.; DeFord, J. H.; Dimayuga, V. L.; Amaning, J. K.; Farrar, R.; Papaconstantinou, J.; Hammers, David W.; Farrar, Roger
Aging and skeletal muscle ischemia/reperfusion (I/R) injury leads to decreased contractile force generation that increases severely with age. Our studies show that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein expression is significantly decreased at 3 and 5 days reperfusion in the young mouse muscle and at 1, 3, 5, and 7 days in the aged muscle. Using PCR, we have shown that GAPDH mRNA levels in young and old muscle increase at 5 days reperfusion compared to control, suggesting that the protein deficit is not transcriptional. Furthermore, while total tyrosine nitration did not increase in the young muscle, GAPDH nitration increased significantly at 1 and 3 days reperfusion. In contrast, total tyrosine nitration in aged muscle increased significantly at 1, 3, and 5 days of reperfusion, with increases in GAPDH nitration at the same time points. We conclude that GAPDH protein levels decrease following I/R, that this is not transcriptionally mediated, that the aged muscle experiences greater oxidative stress, protein modification and GAPDH degradation, possibly contributing to decreased muscle function. We propose that tyrosine nitration enhances GAPDH degradation following I/R and that the persistent decrease of GAPDH in aged muscle is due to the prolonged increase in oxidative modification in this age group.
Local Correlations in Codon Preferences do not Support a Model of tRNA Recycling
(2014-09) Hussmann, Jeffrey A.; Press, William H.; Hussmann, Jeffrey A.; Press, William H.
It has been proposed that patterns in the usage of synonymous codons provide evidence that individual tRNA molecules are recycled through the ribosome, translating several occurrences of the same amino acid before diffusing away. The claimed evidence is based on counting the frequency with which pairs of synonymous codons are used at nearby occurrences of the same amino acid, as compared to the frequency expected if each codon were chosen independently from a single genome-wide distribution. We show that such statistics simply measure variation in codon preferences across a genome. As a negative control on the potential contribution of pressure to exploit tRNA recycling on these signals, we examine correlations in the usage of codons that encode different amino acids. We find that these controls are statistically as strong as the claimed evidence and conclude that there is no informatic evidence that tRNA recycling is a force shaping codon usage.
Membrane-Mediated Alterations of Intracellular Na+ and K+ in Lytic-Virus-Infected and Retrovirus-Transformed Cells
(1982_07) Garry, Robert F.; Ulug, Emin T.; Bose, Henry R.; Garry, Robert F.; Ulug, Emin T.; Bose, Henry R.
Infection of chick-embryo fibroblasts and other ceils by certain animal viruses results in alterations in the intracellular concentrations of Na + and K +. Dramatic alterations in monovalent-cation concentrations of lytic-virus-infected ceils may favor the synthesis of viral proteins over cellular proteins. More subtle alterations in retrovirus-transformed cells may result in the expression of many morphological and biochemical changes associated with the transformed phenotype.
Membrane-Potential of Rat Hepatoma-Cells in Culture - Influence of Factors Affecting Amino-Acid-Transport
(1995-08) Mouat, M. F.; Cantrell, A. C.; Manchester, K. L.; Mouat, M. F.
The effect has been studied of various media, hormones and of amino acids on the membrane potential of rat hepatoma cells in culture measured by microelectrode impalement. Cells in Eagle's minimal essential medium plus 5% serum had a value which varied daily from about 5-8 mV, inside negative. The membrane potential of rat hepatocytes was measured to be 8.7+/-0.2mV, inside negative. The membrane potential of the hepatoma cells was decreased by insulin and increased by glucagon. Membrane potential was unaffected by change of medium to Hanks' or Earle's balanced salt solutions or deprivation of serum. It was, however, reduced in cells in phosphate-buffered saline and by reduction of pH. The former effect was shown to be due to the higher [Na+] of phosphate-buffered saline as opposed to the other media. Addition of alanine, glycine, serine, proline and methylaminoisobutyrate all reduced membrane potential by 2-3 mV. Smaller decreases were seen with methionine, leucine and phenylalanine, but none with glutamine, threonine, BCH (2-aminonorborane-2-carboxylic acid) and D-alanine. The results are compared with the effects of similar conditions on aminoisobutyrate uptake. Whilst there was a correlation under some conditions there was not under others. It is concluded that for the hepatoma cells factors additional to the membrane potential must exert some influence on the capacity for amino acid transport.
Molecular Identification of t(w5): Vps52 Promotes Pluripotential Cell Differentiation Through Cell-Cell Interactions
(2012-11) Sugimoto, Michihiko; Kondo, Masayo; Hirose, Michiko; Suzuki, Misao; Mekada, Kazuyuki; Abe, Takaya; Kiyonari, Hiroshi; Ogura, Atsuo; Takagi, Nobuo; Artzt, Karen; Abe, Kuniya; Artzt, Karen
After implantation, pluripotent epiblasts are converted to embryonic ectoderm through cell-cell interactions that significantly change the transcriptional and epigenetic networks. An entree to understanding this vital developmental transition is the t(w5) mutation of the mouse t complex. This mutation produces highly specific defects in the embryonic ectoderm before gastrulation, leading to death of the embryonic ectoderm. Using a positional cloning approach, we have now identified the mutated gene, completing a decades-long search. The gene, vacuolar protein sorting 52 (Vps52), is a mouse homolog of yeast VPS52 that is involved in the retrograde trafficking of endosomes. Our data suggest that Vps52 acts in extraembryonic tissues to support the growth and differentiation of embryonic ectoderm via cell-cell interactions. It is also required in the formation of embryonic structures at a later stage of development, revealing hitherto unknown functions of Vps52 in the development of a multicellular organism.
Short Inverted Repeats are Hotspots for Genetic Instability: Relevance to Cancer Genomes
(2015-03) Lu, Steve; Wang, Guilang L.; Bacolla, Albino; Zhao, Junhua; Spitser, Scott; Vasquez, Karen M.; Lu, Steve; Wang, Guilang L.; Bacolla, Albino; Zhao, Junhua; Spitser, Scott; Vasquez, Karen M.
Analyses of chromosomal aberrations in human genetic disorders have revealed that inverted repeat sequences (IRs) often co-localize with endogenous chromosomal instability and breakage hotspots. Approximately 80% of all IRs in the human genome are short (<100 bp), yet the mutagenic potential of such short cruciform-forming sequences has not been characterized. Here, we find that short IRs are enriched at translocation breakpoints in human cancer and stimulate the formation of DNA double-strand breaks (DSBs) and deletions in mammalian and yeast cells. We provide evidence for replication-related mechanisms of IR-induced genetic instability and a novel XPF cleavage-based mechanism independent of DNA replication. These discoveries implicate short IRs as endogenous sources of DNA breakage involved in disease etiology and suggest that these repeats represent a feature of genome plasticity that may contribute to the evolution of the human genome by providing a means for diversity within the population.
Tgif1 Counterbalances The Activity Of Core Pluripotency Factors In Mouse Embryonic Stem Cells
(2015-10) Lee, Bum-Kyu; Shen, Wenwen; Lee, Jiwoon; Rhee, Catherine; Chung, Haewon; Kim, Kun-Yong; Park, In-Hyun; Kim, Jonghwan; Lee, Bum-Kyu; Shen, Wenwen; Lee, Jiwoon; Rhee, Catherine; Chung, Haewon; Kim, Jonghwan
Core pluripotency factors, such as Oct4, Sox2, and Nanog, play important roles in maintaining embryonic stem cell (ESC) identity by autoregulatory feedforward loops. Nevertheless, the mechanism that provides precise control of the levels of the ESC core factors without indefinite amplification has remained elusive. Here, we report the direct repression of core pluripotency factors by Tgif1, a previously known terminal repressor of TGF beta/activin/nodal signaling. Overexpression of Tgif1 reduces the levels of ESC core factors, whereas its depletion leads to the induction of the pluripotency factors. We confirm the existence of physical associations between Tgif1 and Oct4, Nanog, and HDAC1/2 and further show the level of Tgif1 is not significantly altered by treatment with an activator/inhibitor of the TGF beta/activin/nodal signaling. Collectively, our findings establish Tgif1 as an integral member of the core regulatory circuitry of mouse ESCs that counterbalances the levels of the core pluripotency factors in a TGF beta/activin/nodal-independent manner.
Transcription Restores DNA Repair to Heterochromatin, Determining Regional Mutation Rates in Cancer Genomes
(2014-11) Zheng, Christina L.; Wang, Nicholas J.; Chung, Jongsuk; Moslehi, Homayoun; Sanborn, J. Zachary; Hur, Joseph S.; Collisson, Eric A.; Vemula, Swapna S.; Naujokas, Agne; Chiotti, Kami E.; Cheng, Jeffrey B.; Fassihi, Hiva; Blumberg, Andrew J.; Bailey, Celeste V.; Fudem, Gary M.; Mihm, Frederick G.; Cunningham, Bari B.; Neuhaus, Isaac M.; Liao, Wilson; Oh, Dennis H.; Cleaver, James E.; LeBoit, Philip E.; Costello, Joseph F.; Lehmann, Alan R.; Gray, Joe W.; Spellman, Paul T.; Arron, Sarah T.; Huh, Nam; Purdom, Elizabeth; Cho, Raymond J.; Blumberg, Andrew J.
Somatic mutations in cancer are more frequent in heterochromatic and late-replicating regions of the genome. We report that regional disparities in mutation density are virtually abolished within transcriptionally silent genomic regions of cutaneous squamous cell carcinomas (cSCCs) arising in an XPC-/- background. XPC-/- cells lack global genome nucleotide excision repair (GG-NER), thus establishing differential access of DNA repair machinery within chromatin-rich regions of the genome as the primary cause for the regional disparity. Strikingly, we find that increasing levels of transcription reduce mutation prevalence on both strands of gene bodies embedded within H3K9me3-dense regions, and only to those levels observed in H3K9me3-sparse regions, also in an XPC-dependent manner. Therefore, transcription appears to reduce mutation prevalence specifically by relieving the constraints imposed by chromatin structure on DNA repair. We model this relationship among transcription, chromatin state, and DNA repair, revealing a new, personalized determinant of cancer risk.