Myosin Va binding to neurofilaments is essential for correct myosin Va distribution and transport and neurofilament density

The identification of molecular motors that modulate the neuronal cytoskeleton has been elusive. Here, we show that a molecular motor protein, myosin Va, is present in high proportions in the cytoskeleton of mouse CNS and peripheral nerves. Immunoelectron microscopy, coimmunoprecipitation, and blot overlay analyses demonstrate that myosin Va in axons associates with neurofilaments, and that the NF-L subunit is its major ligand. A physiological association is indicated by observations that the level of myosin Va is reduced in axons of NF-L-null mice lacking neurofilaments and increased in mice overexpressing NF-L, but unchanged in NF-H-null mice. In vivo pulse-labeled myosin Va advances along axons at slow transport rates overlapping with those of neurofilament proteins and actin, both of which coimmunoprecipitate with myosin Va. Eliminating neurofilaments from mice selectively accelerates myosin Va translocation and redistributes myosin Va to the actin-rich subaxolemma and membranous organelles. Finally, peripheral axons of dilute-lethal mice, lacking functional myosin Va, display selectively increased neurofilament number and levels of neurofilament proteins without altering axon caliber. These results identify myosin Va as a neurofilament-associated protein, and show that this association is essential to establish the normal distribution, axonal transport, and content of myosin Va, and the proper numbers of neurofilaments in axons.     

Gene replacement in mice reveals that the heavily phosphorylated tail of neurofilament heavy subunit does not affect axonal caliber or the transit of cargoes in slow axonal transport

The COOH-terminal tail of mammalian neurofilament heavy subunit (NF-H), the largest neurofilament subunit, contains 44-51 lysine-serine-proline repeats that are nearly stoichiometrically phosphorylated after assembly into neurofilaments in axons. Phosphorylation of these repeats has been implicated in promotion of radial growth of axons, control of nearest neighbor distances between neurofilaments or from neurofilaments to other structural components in axons, and as a determinant of slow axonal transport. These roles have now been tested through analysis of mice in which the NF-H gene was replaced by one deleted in the NF-H tail. Loss of the NF-H tail and all of its phosphorylation sites does not affect the number of neurofilaments, alter the ratios of the three neurofilament subunits, or affect the number of microtubules in axons. Additionally, it does not reduce interfilament spacing of most neurofilaments, the speed of action potential propagation, or mature cross-sectional areas of large motor or sensory axons, although its absence slows the speed of acquisition of normal diameters. Most surprisingly, at least in optic nerve axons, loss of the NF-H tail does not affect the rate of transport of neurofilament subunits.     

FtsH is involved in the early stages of repair of photosystem II in Synechocystis sp PCC 6803

When plants, algae, and cyanobacteria are exposed to excessive light, especially in combination with other environmental stress conditions such as extreme temperatures, their photosynthetic performance declines. A major cause of this photoinhibition is the light-induced irreversible photodamage to the photosystem II (PSII) complex responsible for photosynthetic oxygen evolution. A repair cycle operates to selectively replace a damaged D1 subunit within PSII with a newly synthesized copy followed by the light-driven reactivation of the complex. Net loss of PSII activity occurs (photoinhibition) when the rate of damage exceeds the rate of repair. The identities of the chaperones and proteases involved in the replacement of D1 in vivo remain uncertain. Here, we show that one of the four members of the FtsH family of proteases (cyanobase designation slr0228) found in the cyanobacterium Synechocystis sp PCC 6803 is important for the repair of PSII and is vital for preventing chronic photoinhibition. Therefore, the ftsH gene family is not functionally redundant with respect to the repair of PSII in this organism. Our data also indicate that FtsH binds directly to PSII, is involved in the early steps of D1 degradation, and is not restricted to the removal of D1 fragments. These results, together with the recent analysis of ftsH mutants of Arabidopsis, highlight the critical role played by FtsH proteases in the removal of damaged D1 from the membrane and the maintenance of PSII activity in vivo.     

Structure, stability and function of RNA pseudoknots involved in stimulating ribosomal frameshifting

Programmed -1 ribosomal frameshifting has become the subject of increasing interest over the last several years, due in part to the ubiquitous nature of this translational recoding mechanism in pathogenic animal and plant viruses. All cis-acting frameshift signals encoded in mRNAs are minimally composed of two functional elements: a heptanucleotide "slippery sequence" conforming to the general form X XXY YYZ, followed by an RNA structural element, usually an H-type RNA pseudoknot, positioned an optimal number of nucleotides (5 to 9) downstream. The slippery sequence itself promotes a low level ( approximately 1 %) of frameshifting; however, downstream pseudoknots stimulate this process significantly, in some cases up to 30 to 50 %. Although the precise molecular mechanism of stimulation of frameshifting remains poorly understood, significant advances have been made in our knowledge of the three-dimensional structures, thermodynamics of folding, and functional determinants of stimulatory RNA pseudoknots derived from the study of several well-characterized frameshift signals. These studies are summarized here and provide new insights into the structural requirements and mechanism of programmed -1 ribosomal frameshifting.     

Mutation analysis and embryonic expression of the HLXB9 Currarino syndrome gene

The HLXB9 homeobox gene was recently identified as a locus for autosomal dominant Currarino syndrome, also known as hereditary sacral agenesis (HSA). This gene specifies a 403-amino acid protein containing a homeodomain preceded by a very highly conserved 82-amino acid domain of unknown function; the remainder of the protein is not well conserved. Here we report an extensive mutation survey that has identified mutations in the HLXB9 gene in 20 of 21 patients tested with familial Currarino syndrome. Mutations were also detected in two of seven sporadic Currarino syndrome patients; the remainder could be explained by undetected mosaicism for an HLXB9 mutation or by genetic heterogeneity in the sporadic patients. Of the mutations identified in the 22 index patients, 19 were intragenic and included 11 mutations that could lead to the introduction of a premature termination codon. The other eight mutations were missense mutations that were significantly clustered in the homeodomain, resulting, in each patient, in nonconservative substitution of a highly conserved amino acid. All of the intragenic mutations were associated with comparable phenotypes. The only genotype-phenotype correlation appeared to be the occurrence of developmental delay in the case of three patients with microdeletions. HLXB9 expression was analyzed during early human development in a period spanning Carnegie stages 12-21. Signal was detected in the basal plate of the spinal cord and hindbrain and in the pharynx, esophagus, stomach, and pancreas. Significant spatial and temporal expression differences were evident when compared with expression of the mouse Hlxb9 gene, which may partly explain the significant human-mouse differences in mutant phenotype.     

Expression and distribution of InsP(3) receptor subtypes in proliferating vascular smooth muscle cells

The expression and distribution of types 1, 2, and 3 inositol 1,4, 5-trisphosphate receptor (InsP(3)R) in proliferating, primary cultures of rat aortic smooth muscle were compared to fully developed and differentiated rat aortic smooth muscle. Subtype-specific InsP(3)R antibodies revealed that the expression of type 1 InsP(3)R was similar in cultured aortic cells and aorta homogenate but expression of type 2 and 3 InsP(3)R subtypes was increased 3-fold in cultured aortic cells. The distribution of the type 1 InsP(3)R was located throughout the cytoplasm; type 2 InsP(3)R was found closely associated with the nucleus and at the plasma membrane; type 3 InsP(3)R was distributed predominantly around the nucleus. Alterations in InsP(3)R subtype expression and localization may have important functions in regulating intracellular calcium release around the nucleus when vascular smooth muscle cells switch to a more proliferating phenotype.     

Evaluation of a new apolipoprotein(a) isoform-independent assay for serum Lipoprotein(a)

The risk factor, Lipoprotein(a), [(Lp(a)], has been measured in numerous clinical studies by a variety of immunochemical assay methods. It is becoming apparent that for many of these assays antibody specificity towards the apolipoprotein(a) [apo(a)] repetitive component [the kringle 4 - type 2 repeats] and apo(a) size heterogeneity can significantly affect the accuracy of serum Lp(a) measurements. To address this issue, we investigated whether our current in house Lp(a) [Mercodia] assay showed such bias compared to a recently available assay [Apo-Tek], claiming to possess superior capability for isoform-independent measurement of Lp(a). Levels of Lipoprotein(a) by both Apo-Tek and Mercodia assays correlated inversely with apo(a) isoform sizes. No significant differences were observed between assays in ranges of Lp(a) concentration within each isoform group. The Mercodia assay exhibited similar isoform-independent behaviour to that of Apo-Tek for e quantitation of serum Lipoprotein(a). Essentially identical results were obtained by the two methods, suggesting that Mercodia assay's capture monoclonal antibody also (as is the case for Apo-Tek) does not recognize the kringle 4-type 2 repetitive domain of apo(a). Correlation of Lp(a) concentrations in patient specimens between Apo-Tek and Mercodia assays showed good agreement, although an overall higher degree of imprecision and non-linearity was noted for the Apo-Tek procedure. A change-over to the Apo-Tek assay would therefore not improve on our current assessment of risk contribution from Lp(a) for atherosclerotic vascular disease in individuals with measurable levels of circulating Lipoprotein(a).     

Why do cytotoxic T lymphocytes fail to eliminate hepatitis C virus? Lessons from studies using major histocompatibility complex class I peptide tetramers.

Hepatitis C virus (HCV) infection is a major public health problem, affecting an estimated 3% of the world's population, and over 10% in some countries. Infection in most cases becomes persistent, and can lead to hepatic inflammation, fibrosis and liver failure. The T lymphocyte reponse, in particular that mediated by cytotoxic T lymphocytes (CTLs), is likely to be involved in determining the outcome of infection, although its overall role is not clear. The use of major histocompatibility complex (MHC) class I peptide tetrameric complexes (tetramers) to study antiviral CTL responses has revolutionized our approach to the study of human infection. We have used a panel of MHC class I tetramers to analyse immune responses in HCV-infected individuals at various stages of disease. We find that the CTL response against HCV is vigorous in its early phases but dwindles over time both in terms of lymphocyte number and function. A number of potential explanations for this 'CTL failure' are discussed.