scholarly journals Centractin (ARP1) associates with spectrin revealing a potential mechanism to link dynactin to intracellular organelles.

1996 ◽  
Vol 135 (6) ◽  
pp. 1815-1829 ◽  
Author(s):  
E A Holleran ◽  
M K Tokito ◽  
S Karki ◽  
E L Holzbaur
Keyword(s):  
Affinity Chromatography ◽  
Mammalian Cells ◽  
Cytoskeletal Proteins ◽  
Transfected Cells ◽  
Cytoskeletal Network ◽  
Biochemical Assays ◽  
Marker Proteins ◽  
Erythroid Spectrin ◽  
Bona Fide ◽  
Dynactin Complex

Centractin (Arp1), an actin-related protein, is a component of the dynactin complex. To investigate potential functions of the protein, we used transient transfections to overexpress centractin in mammalian cells. We observed that the overexpressed polypeptide formed filamentous structures that were significantly longer and more variable in length than those observed in the native dynactin complex. The centractin filaments were distinct from conventional actin in subunit composition and pharmacology as demonstrated by the absence of immunoreactivity of these filaments with an actin-specific antibody, by resistance to treatment with the drug cytochalasin D, and by the inability to bind phalloidin. We examined the transfected cells for evidence of specific associations of the novel centractin filaments with cellular organelles or cytoskeletal proteins. Using immunocytochemistry we observed the colocalization of Golgi marker proteins with the centractin polymers. Additional immunocytochemical analysis using antibodies to non-erythroid spectrin (fodrin) and Golgi-spectrin (beta I sigma *) revealed that spectrin colocalized with the centractin filaments in transfected cells. Biochemical assays demonstrated that spectrin was present in dynactin-enriched cellular fractions, was coimmunoprecipitated from rat brain cytosol using antibodies to dynactin subunits, and was coeluted with dynactin using affinity chromatography. Immunoprecipitations and affinity chromatography also revealed that actin is not a bona fide component of dynactin. Our results indicate that spectrin is associated with the dynactin complex. We suggest a model in which dynactin associates with the Golgi through an interaction between the centractin filament of the dynactin complex and a spectrin-linked cytoskeletal network.

Dissection of the molecular mechanisms of protein targeting to the peroxisome using immunofluorescence and immunocryoelectron microscopies

1990 ◽  
Vol 48 (3) ◽  
pp. 44-45
Author(s):  
G-A. Keller ◽  
S. J. Gould ◽  
S. Subramani ◽  
S. Krisans
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Protein Targeting ◽  
Firefly Luciferase ◽  
Peroxisomal Enzyme ◽  
Transfected Cells ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Series Of Experiments ◽  
Peroxisomal Targeting Signal

Subcellular compartments within eukaryotic cells must each be supplied with unique sets of proteins that must be directed to, and translocated across one or more membranes of the target organelles. This transport is mediated by cis- acting targeting signals present within the imported proteins. The following is a chronological account of a series of experiments designed and carried out in an effort to understand how proteins are targeted to the peroxisomal compartment.-We demonstrated by immunocryoelectron microscopy that the enzyme luciferase is a peroxisomal enzyme in the firefly lantern. -We expressed the cDNA encoding firefly luciferase in mammalian cells and demonstrated by immunofluorescence that the enzyme was transported into the peroxisomes of the transfected cells. -Using deletions, linker insertions, and gene fusion to identify regions of luciferase involved in its transport to the peroxisomes, we demonstrated that luciferase contains a peroxisomal targeting signal (PTS) within its COOH-terminal twelve amino acid.

scholarly journals An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III.

1994 ◽  
Vol 14 (1) ◽  
pp. 68-76 ◽  
Author(s):  
K W Caldecott ◽  
C K McKeown ◽  
J D Tucker ◽  
S Ljungquist ◽  
L H Thompson
Keyword(s):  
Dna Repair ◽  
Affinity Chromatography ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Affinity Purification ◽  
Alkylating Agents ◽  
Chinese Hamster ◽  
Dna Ligase ◽  
Base Excision ◽  
Dna Ligase Iii

XRCC1, the human gene that fully corrects the Chinese hamster ovary DNA repair mutant EM9, encodes a protein involved in the rejoining of DNA single-strand breaks that arise following treatment with alkylating agents or ionizing radiation. In this study, a cDNA minigene encoding oligohistidine-tagged XRCC1 was constructed to facilitate affinity purification of the recombinant protein. This construct, designated pcD2EHX, fully corrected the EM9 phenotype of high sister chromatid exchange, indicating that the histidine tag was not detrimental to XRCC1 activity. Affinity chromatography of extract from EM9 cells transfected with pcD2EHX resulted in the copurification of histidine-tagged XRCC1 and DNA ligase III activity. Neither XRCC1 or DNA ligase III activity was purified during affinity chromatography of extract from EM9 cells transfected with pcD2EX, a cDNA minigene that encodes untagged XRCC1, or extract from wild-type AA8 or untransfected EM9 cells. The copurification of DNA ligase III activity with histidine-tagged XRCC1 suggests that the two proteins are present in the cell as a complex. Furthermore, DNA ligase III activity was present at lower levels in EM9 cells than in AA8 cells and was returned to normal levels in EM9 cells transfected with pcD2EHX or pcD2EX. These findings indicate that XRCC1 is required for normal levels of DNA ligase III activity, and they implicate a major role for this DNA ligase in DNA base excision repair in mammalian cells.

scholarly journals New perspectives on cytoskeletal dysregulation and mitochondrial mislocalization in amyotrophic lateral sclerosis

2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Frances Theunissen ◽  
Phillip K. West ◽  
Samuel Brennan ◽  
Bojan Petrović ◽  
Kosar Hooshmand ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Motor Neurons ◽  
Retrograde Transport ◽  
Cytoskeletal Proteins ◽  
Charcot Marie Tooth Disease ◽  
Mitochondrial Homeostasis ◽  
Axonal Projections ◽  
Cytoskeletal Network ◽  
Brain And Spinal Cord ◽  
Lateral Sclerosis

AbstractAmyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective, early degeneration of motor neurons in the brain and spinal cord. Motor neurons have long axonal projections, which rely on the integrity of neuronal cytoskeleton and mitochondria to regulate energy requirements for maintaining axonal stability, anterograde and retrograde transport, and signaling between neurons. The formation of protein aggregates which contain cytoskeletal proteins, and mitochondrial dysfunction both have devastating effects on the function of neurons and are shared pathological features across several neurodegenerative conditions, including ALS, Alzheimer's disease, Parkinson's disease, Huntington’s disease and Charcot-Marie-Tooth disease. Furthermore, it is becoming increasingly clear that cytoskeletal integrity and mitochondrial function are intricately linked. Therefore, dysregulations of the cytoskeletal network and mitochondrial homeostasis and localization, may be common pathways in the initial steps of neurodegeneration. Here we review and discuss known contributors, including variants in genetic loci and aberrant protein activities, which modify cytoskeletal integrity, axonal transport and mitochondrial localization in ALS and have overlapping features with other neurodegenerative diseases. Additionally, we explore some emerging pathways that may contribute to this disruption in ALS.

scholarly journals Dynamin-related Protein Drp1 Is Required for Mitochondrial Division in Mammalian Cells

2001 ◽  
Vol 12 (8) ◽  
pp. 2245-2256 ◽  
Author(s):  
Elena Smirnova ◽  
Lorena Griparic ◽  
Dixie-Lee Shurland ◽  
Alexander M. van der Bliek
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Subcellular Fractionation ◽  
Related Protein ◽  
Direct Role ◽  
Mitochondrial Division ◽  
Transfected Cells ◽  
Green Fluorescent ◽  
Time Lapse Photography

Mutations in the human dynamin-related protein Drp1 cause mitochondria to form perinuclear clusters. We show here that these mitochondrial clusters consist of highly interconnected mitochondrial tubules. The increased connectivity between mitochondria indicates that the balance between mitochondrial division and fusion is shifted toward fusion. Such a shift is consistent with a block in mitochondrial division. Immunofluorescence and subcellular fractionation show that endogenous Drp1 is localized to mitochondria, which is also consistent with a role in mitochondrial division. A direct role in mitochondrial division is suggested by time-lapse photography of transfected cells, in which green fluorescent protein fused to Drp1 is concentrated in spots that mark actual mitochondrial division events. We find that purified human Drp1 can self-assemble into multimeric ring-like structures with dimensions similar to those of dynamin multimers. The structural and functional similarities between dynamin and Drp1 suggest that Drp1 wraps around the constriction points of dividing mitochondria, analogous to dynamin collars at the necks of budding vesicles. We conclude that Drp1 contributes to mitochondrial division in mammalian cells.

Polyomavirus-plasmid recombinants capable of replicating have an enhanced transforming potential

1983 ◽  
Vol 3 (9) ◽  
pp. 1670-1674
Author(s):  
W J Muller ◽  
M A Naujokas ◽  
J A Hassell
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
T Antigen ◽  
Viral Gene ◽  
Large T Antigen ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Transfected Cells ◽  
Cis Acting ◽  
Viral Gene Product

The frequency of transformation of rodent fibroblasts by polyomavirus is enhanced by a viral gene product, large T-antigen. However, this effect of large T-antigen cannot be demonstrated with pBR322-cloned viral DNA. Recently, it was discovered that pBR322 contains cis-acting sequences inhibitory to DNA replication in mammalian cells. Because polyomavirus large T-antigen is required for viral DNA replication, we examined the possibility that our inability to demonstrate a requirement for large T-antigen in transformation with pBR322-cloned viral DNA was due to the failure of the chimeric DNA to replicate in the transfected cells. To this end we constructed polyomavirus recombinant molecules with a plasmid (pML-2) that lacks these "poison" sequences and measured their capacity to transform cells. Here we report that recombinant plasmids capable of replicating in the transfected cells transform these cells at frequencies approximately sixfold greater than their replication-defective counterparts.

scholarly journals C4orf41 and TTC-15 are mammalian TRAPP components with a role at an early stage in ER-to-Golgi trafficking

2011 ◽  
Vol 22 (12) ◽  
pp. 2083-2093 ◽  
Author(s):  
P. James Scrivens ◽  
Baraa Noueihed ◽  
Nassim Shahrzad ◽  
Sokunthear Hul ◽  
Stephanie Brunet ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Multidisciplinary Approach ◽  
Early Stage ◽  
Binary Interaction ◽  
The Novel ◽  
Novel Proteins ◽  
Bona Fide ◽  
Interaction Map ◽  
Equivalent Complex

TRAPP is a multisubunit tethering complex implicated in multiple vesicle trafficking steps in Saccharomyces cerevisiae and conserved throughout eukarya, including humans. Here we confirm the role of TRAPPC2L as a stable component of mammalian TRAPP and report the identification of four novel components of the complex: C4orf41, TTC-15, KIAA1012, and Bet3L. Two of the components, KIAA1012 and Bet3L, are mammalian homologues of Trs85p and Bet3p, respectively. The remaining two novel TRAPP components, C4orf41 and TTC-15, have no homologues in S. cerevisiae. With this work, human homologues of all the S. cerevisiae TRAPP proteins, with the exception of the Saccharomycotina-specific subunit Trs65p, have now been reported. Through a multidisciplinary approach, we demonstrate that the novel proteins are bona fide components of human TRAPP and implicate C4orf41 and TTC-15 (which we call TRAPPC11 and TRAPPC12, respectively) in ER-to-Golgi trafficking at a very early stage. We further present a binary interaction map for all known mammalian TRAPP components and evidence that TRAPP oligomerizes. Our data are consistent with the absence of a TRAPP I–equivalent complex in mammalian cells, suggesting that the fundamental unit of mammalian TRAPP is distinct from that characterized in S. cerevisiae.

scholarly journals Role of Ced-3/ICE-family proteases in staurosporine-induced programmed cell death.

1996 ◽  
Vol 133 (5) ◽  
pp. 1041-1051 ◽  
Author(s):  
M D Jacobsen ◽  
M Weil ◽  
M C Raff
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Mammalian Cell ◽  
Mammalian Cells ◽  
Apoptotic Cell ◽  
Interleukin 1 ◽  
Cell Types ◽  
Cysteine Proteases ◽  
A Cell ◽  
Bona Fide

In the accompanying paper by Weil et al. (1996) we show that staurosporine (STS), in the presence of cycloheximide (CHX) to inhibit protein synthesis, induces apoptotic cell death in a large variety of nucleated mammalian cell types, suggesting that all nucleated mammalian cells constitutively express all of the proteins required to undergo programmed cell death (PCD). The reliability of that conclusion depends on the evidence that STS-induced, and (STS + CHS)-induced, cell deaths are bona fide examples of PCD. There is rapidly accumulating evidence that some members of the Ced-3/Interleukin-1 beta converting enzyme (ICE) family of cysteine proteases are part of the basic machinery of PCD. Here we show that Z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a cell-permeable, irreversible, tripeptide inhibitor of some of these proteases, suppresses STS-induced and (STS + CHX)-induced cell death in a wide variety of mammalian cell types, including anucleate cytoplasts, providing strong evidence that these are all bona fide examples of PCD. We show that the Ced-3/ICE family member CPP32 becomes activated in STS-induced PCD, and that Bcl-2 inhibits this activation. Most important, we show that, in some cells at least, one or more CPP32-family members, but not ICE itself, is required for STS-induced PCD. Finally, we show that zVAD-fmk suppresses PCD in the interdigital webs in developing mouse paws and blocks the removal of web tissue during digit development, suggesting that this inhibition will be a useful tool for investigating the roles of PCD in various developmental processes.

scholarly journals Proteomic analysis of high NaCl-induced changes in abundance of nuclear proteins

2012 ◽  
Vol 44 (21) ◽  
pp. 1063-1071 ◽  
Author(s):  
Jinxi Li ◽  
Joan D. Ferraris ◽  
Danni Yu ◽  
Taruna Singh ◽  
Yuichiro Izumi ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Nuclear Protein ◽  
Nuclear Translocation ◽  
Biological Significance ◽  
Renal Medulla ◽  
Cytoskeletal Proteins ◽  
Hek293 Cells ◽  
Protein Abundance ◽  
Dynamic Changes ◽  
Cytoskeleton Organization

Mammalian cells are normally stressed by high interstitial NaCl in the renal medulla and by lesser elevation of NaCl in several other tissues. High NaCl damages proteins and DNA and can kill cells. Known protective responses include nuclear translocation of the transcription factor NFAT5 and other proteins. In order better to understand the extent and significance of changes in nuclear protein abundance, we extracted nuclear and cytoplasmic proteins separately from HEK293 cells and measured by LC-MS/MS (iTRAQ) changes of abundance of proteins in the extracts in response to high NaCl at three time points: 1 h, 8 h, and adapted for two passages. We confidently identified a total of 3,190 proteins; 163 proteins changed significantly at least at one time point in the nucleus. We discerned the biological significance of the changes by Gene Ontology and protein network analysis. Proteins that change in the nucleus include ones involved in protein folding and localization, microtubule-based process, regulation of cell death, cytoskeleton organization, DNA metabolic process, RNA processing, and cell cycle. Among striking changes in the nucleus, we found a decrease of all six 14-3-3 isoforms; dynamic changes of “cytoskeletal” proteins, suggestive of nucleoskeletal reorganization; rapid decrease of tubulins; and dynamic changes of heat shock proteins. Identification of these changes of nuclear protein abundance enhances our understanding of high NaCl-induced cellular stress, and provides leads to previously unknown damages and protective responses.

scholarly journals Polyomavirus-plasmid recombinants capable of replicating have an enhanced transforming potential.

1983 ◽  
Vol 3 (9) ◽  
pp. 1670-1674 ◽  
Author(s):  
W J Muller ◽  
M A Naujokas ◽  
J A Hassell
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
T Antigen ◽  
Viral Gene ◽  
Large T Antigen ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Transfected Cells ◽  
Cis Acting ◽  
Viral Gene Product

The frequency of transformation of rodent fibroblasts by polyomavirus is enhanced by a viral gene product, large T-antigen. However, this effect of large T-antigen cannot be demonstrated with pBR322-cloned viral DNA. Recently, it was discovered that pBR322 contains cis-acting sequences inhibitory to DNA replication in mammalian cells. Because polyomavirus large T-antigen is required for viral DNA replication, we examined the possibility that our inability to demonstrate a requirement for large T-antigen in transformation with pBR322-cloned viral DNA was due to the failure of the chimeric DNA to replicate in the transfected cells. To this end we constructed polyomavirus recombinant molecules with a plasmid (pML-2) that lacks these "poison" sequences and measured their capacity to transform cells. Here we report that recombinant plasmids capable of replicating in the transfected cells transform these cells at frequencies approximately sixfold greater than their replication-defective counterparts.

MVP interacts with YPEL4 and inhibits YPEL4-mediated activities of the ERK signal pathway

2010 ◽  
Vol 88 (3) ◽  
pp. 445-450 ◽  
Author(s):  
Pei Liang ◽  
Yongqi Wan ◽  
Yan Yan ◽  
Yuequn Wang ◽  
Na Luo ◽  
...  
Keyword(s):  
Metal Binding ◽  
Mammalian Cells ◽  
Mapk Signaling ◽  
Reporter System ◽  
Biochemical Assays ◽  
Metal Binding Domain ◽  
Erk Signal Pathway ◽  
Gene Maps ◽  
Putative Zinc Finger ◽  
Two Hybrid

Human YPEL4 is a member of YPEL family. It contains a Yippee domain, which is a putative zinc-finger-like, metal-binding domain. The human YPEL4 gene maps to chromosome 11q12.1, is ubiquitously expressed in adult tissues, and encodes a nuclear protein of 127 amino acids, the function of which remains unknown. To gain insights into the cellular function of this protein, we searched for YPEL4-interacting proteins using a yeast two-hybrid screen. The major vault protein (MVP), a lung resistance associated protein, was identified as a binding partner of YPEL4. The interaction between YPEL4 and MVP in mammalian cells was further demonstrated by a series of biochemical assays including the mammalian two-hybrid assay, GST pull-down assay, co-immunoprecipitation assay, and immunocytochemistry. Using a reporter system, we found that MVP can inhibit YPEL4’s ability to activate Elk-1 in the MAPK signaling pathway. This study provides new clues for understanding the molecular mechanism of YPEL4 in cell division and signal transduction pathways and should be helpful for understanding molecular functions of the YPEL family.

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