Triacylglycerol Synthesis Enzymes Mediate Lipid Droplet Growth by Relocalizing from the ER to Lipid Droplets

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Highlights? Triacylglyceride (TG) synthesis is coupled with lipid droplet (LD) growth ? Two LD populations exist: growing LDs, containing TG enzymes, and small LDs ? Specific TG synthesis enzymes move from the ER to LDs through membrane bridges ? LD localization of TG enzymes mediates expansion of a subset of LDs     

First isolation of an aquatic birnavirus from farmed and wild fish species in Australia

During routine sampling and testing, as part of a systematic surveillance program (the Tasmanian Salmonid Health Surveillance Program), an aquatic birnavirus was isolated from 'pin-head' (fish exhibiting deficient acclimatisation on transfer to saltwater) Atlantic salmon Salmo salar, approximately 18 mo old, farmed in net-pens located in Macquarie Harbour on the west coast of Tasmania, Australia. The isolate grows readily in a range of fish cell lines including CHSE-214, RTG-2 and BF-2 and is neutralised by a pan-specific rabbit antiserum raised against infectious pancreatic necrosis virus (IPNV) Ab strain and by a commercial pan-specific IPNV-neutralising monoclonal antibody. Presence of the virus was not associated with gross clinical signs. Histopathological examination revealed a range of lesions particularly in pancreatic tissue. The virus was localised in pancreas sections by immunoperoxidase staining using the polyclonal antiserum and by electron microscopy. Examination by electron microscopy demonstrated that the virus isolated in cell culture (1) belongs to the family Birnaviridae, genus Aquabirnaviridae; (2) was ultrastructurally and antigenically similar to virus identified in the index fish; (3) is related to IPNV. Western blot analysis using the polyclonal rabbit antiserum confirmed the cross-reactions between various aquatic birnavirus isolates. In addition, PCR analysis of isolated viral nucleic acid from the index case indicated that the virus is more closely related to IPNV fr21 and N1 isolates than to other birnavirus isolates available for comparison. Sampling of other fish species within Macquarie Harbour has demonstrated that the virus is present in several other species of fish including farmed rainbow trout Oncorhynchus mykiss, wild flounder Rhombosolea tapirina, cod Pseudophycis sp., spiked dogfish Squalus megalops and ling Genypterus blacodes.     

Profiles of photosynthesis within red and green leaves of Quintinia serrata

We have measured photosynthesis at the cellular, tissue, and whole leaf levels to understand the role of anthocyanin pigments on patterns of light utilization. Profiles of chlorophyll fluorescence through sections of red and green leaves of Quintinia serrata showed that anthocyanins in the mesophyll restricted absorption of green light to the uppermost palisade mesophyll. The distribution was further restricted when anthocyanins were also present in the upper epidermis. Mesophyll cells located beneath a cyanic light-filter assumed the characteristic photosynthetic features of shade-adapted cells. As a result, red leaves showed a 23% reduction in CO₂ assimilation under light-saturating conditions, and a lower threshold irradiance for light-saturation, relative to those of green leaves. The photosynthetic characteristics of red leaves are comparable to those of shade-acclimated plants.     

Antibodies directed against the peroxisomal targeting signal of firefly luciferase recognize multiple mammalian peroxisomal proteins

We have previously shown that the peroxisomal targeting signal in firefly luciferase consists of the COOH-terminal three amino acids of the protein, serine-lysine-leucine (Gould, S.J., G.A. Keller, N. Hosken, J. Wilkinson, and S. Subramani, 1989. J. Cell Biol. 108:1657-1664). Antibodies were raised against a synthetic peptide that contained this tripeptide at its COOH terminus. Immunofluorescence and immunocryoelectron microscopy revealed that the anti-peptide antibodies specifically detected peroxisomes in mammalian cells. Further characterization revealed that the antibodies were primarily directed against the COOH-terminal three amino acids of the peptide. In Western blot experiments, the antibodies recognized 15-20 rat liver peroxisomal proteins, but reacted with only a few proteins from other subcellular compartments. These results provide independent immunological evidence that the peroxisomal targeting signal identified in firefly luciferase is present in many peroxisomal proteins.     

SmeA, a small membrane protein with multiple functions in Streptomyces sporulation including targeting of a SpoIIIE/FtsK-like protein to cell division septa

Sporulation in aerial hyphae of Streptomyces coelicolor involves profound changes in regulation of fundamental morphogenetic and cell cycle processes to convert the filamentous and multinucleoid cells to small unigenomic spores. Here, a novel sporulation locus consisting of smeA (encoding a small putative membrane protein) and sffA (encoding a SpoIIIE/FtsK-family protein) is characterized. Deletion of smeA-sffA gave rise to pleiotropic effects on spore maturation, and influenced the segregation of chromosomes and placement of septa during sporulation. Both smeA and sffA were expressed specifically in apical cells of sporogenic aerial hyphae simultaneously with or slightly after Z-ring assembly. The presence of smeA-like genes in streptomycete chromosomes, plasmids and transposons, often paired with a gene for a SpoIIIE/FtsK- or Tra-like protein, indicates that SmeA and SffA functions might be related to DNA transfer. During spore development SffA accumulated specifically at sporulation septa where it colocalized with FtsK. However, sffA did not show redundancy with ftsK, and SffA function appeared distinct from the DNA translocase activity displayed by FtsK during closure of sporulation septa. The septal localization of SffA was dependent on SmeA, suggesting that SmeA may act as an assembly factor for SffA and possibly other proteins required during spore maturation.     

Superresolution imaging of multiple fluorescent proteins with highly overlapping emission spectra in living cells

Localization-based superresolution optical imaging is rapidly gaining popularity, yet limited availability of genetically encoded photoactivatable fluorescent probes with distinct emission spectra impedes simultaneous visualization of multiple molecular species in living cells. We introduce PAmKate, a monomeric photoactivatable far-red fluorescent protein, which facilitates simultaneous imaging of three photoactivatable proteins in mammalian cells using fluorescence photoactivation localization microscopy (FPALM). Successful probe identification was achieved by measuring the fluorescence emission intensity in two distinct spectral channels spanning only ∼100 nm of the visible spectrum. Raft-, non-raft-, and cytoskeleton-associated proteins were simultaneously imaged in both live and fixed fibroblasts coexpressing Dendra2-hemagglutinin, PAmKate-transferrin receptor, and PAmCherry1-β-actin fusion constructs, revealing correlations between the membrane proteins and membrane-associated actin structures.     

A transgene carrying an A2G missense mutation in the SMN gene modulates phenotypic severity in mice with severe (type I) spinal muscular atrophy

5q spinal muscular atrophy (SMA) is a common autosomal recessive disorder in humans and the leading genetic cause of infantile death. Patients lack a functional survival of motor neurons (SMN1) gene, but carry one or more copies of the highly homologous SMN2 gene. A homozygous knockout of the single murine Smn gene is embryonic lethal. Here we report that in the absence of the SMN2 gene, a mutant SMN A2G transgene is unable to rescue the embryonic lethality. In its presence, the A2G transgene delays the onset of motor neuron loss, resulting in mice with mild SMA. We suggest that only in the presence of low levels of full-length SMN is the A2G transgene able to form partially functional higher order SMN complexes essential for its functions. Mild SMA mice exhibit motor neuron degeneration, muscle atrophy, and abnormal EMGs. Animals homozygous for the mutant transgene are less severely affected than heterozygotes. This demonstrates the importance of SMN levels in SMA even if the protein is expressed from a mutant allele. Our mild SMA mice will be useful in (a) determining the effect of missense mutations in vivo and in motor neurons and (b) testing potential therapies in SMA.