6 Papers found (0.083 seconds)

Pollard AK et al. (2020) Astrobiology "Molecular Muscle Experiment: Hardware and Operational Lessons for Future Astrobiology Space Experiments."

Vanapalli SA, Balsamo M, Deane CS, Etheridge T, Gaffney CJ, Pollard AK, Szewczyk NJ, Cooke M, Mariani A, Mierzwa BE, Ellwood RA, Hewitt JE

[

Astrobiology,

2020]

Biology experiments in space seek to increase our understanding of what happens to life beyond Earth and how we can safely send life beyond Earth. Spaceflight is associated with many (mal)adaptations in physiology, including decline in musculoskeletal, cardiovascular, vestibular, and immune systems. Biological experiments in space are inherently challenging to implement. Development of hardware and validation of experimental conditions are critical to ensure the collection of high-quality data. The model organism Caenorhabditis elegans has been studied in space for more than 20 years to better understand spaceflight-induced (patho)physiology, particularly spaceflight-induced muscle decline. These experiments have used a variety of hardware configurations. Despite this, hardware used in the past was not available for our most recent experiment, the Molecular Muscle Experiment (MME). Therefore, we had to design and validate flight hardware for MME. MME provides a contemporary example of many of the challenges faced by researchers conducting C. elegans experiments onboard the International Space Station. Here, we describe the hardware selection and validation, in addition to the ground-based experiment scientific validation testing. These experiences and operational solutions allow others to replicate and/or improve our experimental design on future missions.

Pollard AK et al. (2019) Aging (Albany NY) "A comparison of the mitochondrial proteome and lipidome in the mouse and long-lived Pipistrelle bats."

Liddell S, Pollard AK, Shephard F, Brown M, Ingram TL, Ortori CA, Barrett DA, Chakrabarti L

[

Aging (Albany NY),

2019]

It is accepted that smaller mammals with higher metabolic rates have shorter lifespans. The very few species that do not follow these rules can give insights into interesting differences. The recorded maximum lifespans of bats are exceptional - over 40 years, compared with the laboratory mouse of 4 years. We investigated the differences in the biochemical composition of mitochondria between bat and mouse species. We used proteomics and ultra-high-performance liquid chromatography coupled with high resolution mass spectrometry lipidomics, to interrogate mitochondrial fractions prepared from Mus musculus and Pipistrellus pipistrellus brain and skeletal muscle. Fatty acid binding protein 3 was found at different levels in mouse and bat muscle mitochondria and its orthologues were investigated in Caenorhabditis elegans knock-downs for LBP 4, 5 and 6. In the bat, high levels of free fatty acids and N-acylethanolamine lipid species together with a significantly greater abundance of fatty acid binding protein 3 in muscle (1.8-fold, p=0.037) were found. Manipulation of fatty acid binding protein orthologues in C. elegans suggest these proteins and their role in lipid regulation are important for mitochondrial function.

Mohri K et al. (2006) Mol Biol Cell "Enhancement of actin-depolymerizing factor/cofilin-dependent actin disassembly by actin-interacting protein 1 is required for organized actin filament assembly in the Caenorhabditis elegans body wall muscle."

Ono K, Ono S, Yu R, Mohri K, Yamashiro S

[

Mol Biol Cell,

2006]

Monitoring Editor: Thomas Pollard Regulated disassembly of actin filaments is involved in a number of cellular processes that require dynamic rearrangement of the actin cytoskeleton. Actin-interacting protein 1 (AIP1) specifically enhances disassembly of actin depolymerizing factor (ADF)/cofilin-bound actin filaments. In vitro, AIP1 actively disassembles filaments, caps barbed ends, and binds to the side of filaments. However, how AIP1 functions in the cellular actin cytoskeletal dynamics is not understood. We compared biochemical and in vivo activities of mutant UNC-78 proteins and found that impaired activity of mutant UNC-78 proteins to enhance disassembly of ADF/cofilin-bound actin filaments is associated with inability to regulate striated organization of actin filaments in muscle cells. Six functionally important residues are present in the N-terminal beta-propeller, whereas one residue is located in the C-terminal beta-propeller, suggesting the presence of two separate sites for interaction with ADF/cofilin and actin. In vitro, these mutant UNC-78 proteins exhibited variable alterations in actin disassembly and/or barbed-end capping activities, suggesting that both activities are important for its in vivo function. These results indicate that the actin-regulating activity of AIP1 in cooperation with ADF/cofilin is essential for its in vivo function to regulate actin filament organization in muscle cells.

Mercer KB et al. (2006) Mol Biol Cell "Caenorhabditis elegans UNC-96 is a new component of M-lines that interacts with UNC-98 and paramyosin and is required in adult muscle for assembly and/or maintenance of thick filaments."

Tinley TL, Qadota H, Sheth S, Miller RK, Benian GM, Mercer KB

[

Mol Biol Cell,

2006]

Monitoring Editor: Thomas Pollard To gain further insight into the molecular architecture, assembly and maintenance of the sarcomere, we have carried out a molecular analysis of the UNC-96 protein in the muscle of C. elegans. By polarized light microscopy of bodywall muscle, unc-96 mutants display reduced myofibrillar organization and characteristic birefringent "needles". By immunofluorescent staining of known myofibril components, unc-96 mutants show major defects in the organization of M-lines, and in the localization of a major thick filament component, paramyosin. In unc-96 mutants, the birefringent needles, which contain both UNC-98 and paramyosin, can be suppressed by starvation or by exposure to reduced temperature. UNC-96 is a novel approximately 47 kDa polypeptide that has regions of similarity to several vertebrate proteins, but no recognizable domains. Antibodies generated to UNC-96 localize the protein to the M-line, a region of the sarcomere in which thick filaments are cross-linked. By genetic and biochemical criteria, UNC-96 interacts with UNC-98, a previously described component of M-lines, and paramyosin. Additionally, UNC-96 copurifies with native thick filaments. A model is presented in which UNC-96 is required in adult muscle to promote thick filament assembly and/or maintenance.

Ono K et al. (2006) Mol Biol Cell "Caenorhabditis elegans kettin, a large immunoglobulin-like repeat protein, binds to filamentous actin and provides mechanical stability to the contractile apparatuses in body wall muscle."

Ono S, Mohri K, Yu R, Ono K

[

Mol Biol Cell,

2006]

Monitoring Editor: Thomas Pollard Kettin is a large actin-binding protein with immunoglobulin-like (Ig) repeats, which is associated with the thin filaments in arthropod muscles. Here, we report identification and functional characterization of kettin in the nematode Caenorhabditis elegans. We found that one of the monoclonal antibodies that were raised against C. elegans muscle proteins specifically reacts with kettin (Ce-kettin). We determined the entire cDNA sequence of Ce-kettin that encodes a protein of 472 kDa with 31 Ig repeats. Arthropod kettins are splice variants of much larger connectin/titin-related proteins. However, the gene for Ce-kettin is independent of other connectin/titin-related genes. Ce-kettin localizes to the thin filaments near the dense bodies in both striated and nonstriated muscles. The C-terminal four Ig repeats and the adjacent nonIg region synergistically bind to actin filaments in vitro. RNA interference of Ce-kettin caused weak disorganization of the actin filaments in body wall muscle. This phenotype was suppressed by inhibiting muscle contraction by a myosin mutation, but enhanced by tetramisole-induced hypercontraction. Furthermore, Ce-kettin was involved in organizing the cytoplasmic portion of the dense bodies in cooperation with alpha-actinin. These results suggest that kettin is an important regulator of myofibrillar organization and provides mechanical stability to the myofibrils during contraction.

Miller RK et al. (2008) Mol Biol Cell "UNC-98 and UNC-96 interact with paramyosin to promote its incorporation into thick filaments of Caenorhabditis elegans."

Benian GM, Mercer KB, Qadota H, Miller RK, Gernert KM

[

Mol Biol Cell,

2008]

Monitoring Editor: Thomas Pollard Mutations in unc-96 or unc-98 cause reduced motility and a characteristic defect in muscle structure: by polarized light microscopy birefringent needles are found at the ends of muscle cells. Anti-paramyosin stains the needles in unc-96 and unc-98 mutant muscle. However there is no difference in the overall level of paramyosin in wild-type, unc-96 and unc-98 animals. Anti-UNC-98 and anti-paramyosin colocalize in the paramyosin accumulations of missense alleles of unc-15 (encodes paramyosin). Anti-UNC-96 (Mercer et al., 2006) and anti-UNC-98 have diffuse localization within muscles of unc-15 null mutants. By immunoblot, in the absence of paramyosin, UNC-98 is diminished, whereas in paramysoin missense mutants, UNC-98 is increased. unc-98 and unc-15, or unc-96 and unc-15 (Mercer et al., 2006) interact genetically either as double heterozygotes or as double homozygotes. By yeast 2-hybrid and ELISAs using purified proteins, UNC-98 interacts with paramyosin residues 31-693, whereas UNC-96 interacts with a separate region of paramyosin, residues 699-798. The importance of surface charge of this 99 residue region for UNC-96 binding was shown. Paramyosin lacking the C terminal UNC-96 binding region fails to localize throughout A-bands. We propose a model in which UNC-98 and UNC-96 may act as chaperones to promote the incorporation of paramyosin into thick filaments.