Biomedical subjects

R B Cary

Publications and source records attributed to R B Cary.

6 recordsLinked to original sources

PubMed1995

Disruption of intermediate filament organization leads to structural defects at the intersomite junction in Xenopus myotomal muscle.

In mature striated muscle, intermediate filaments (IFs) are associated with the periphery of Z-discs and sites of myofibril-membrane attachment. Previously T. Schultheiss, Z. X. Lin, H. Ishikawa, I. Zamir, C. J. Stoeckert and H. Holtzer (1991) J. Cell Biol. 114, 953) reported that the disruption of IF organization in cultured chick myotubes had no detectable effect on muscle cell structure. Cultured muscle is not, however, under the mechanical loads characteristic of muscle in situ. The dorsal myotomal muscle (DMM) of the Xenopus tadpole provides an accessible model system in which to study the effects of mutant IF proteins on an intact, functional muscle. DNAs encoding truncated forms of Xenopus vimentin or desmin were injected into fertilized Xenopus eggs. Embryos were allowed to develop to the tadpole stage and then examined by confocal or electron microscopy. DMM cells containing the truncated IF polypeptides displayed disorganized IF systems. While the alignment of Z-lines appeared unaffected, cells accumulating mutant IF polypeptides displayed abnormal organization at the intersomite junction. Myocyte termini are normally characterized by deep invaginations of the sarcolemma. In myocytes expressing mutated IF polypeptides, these membrane invaginations were reduced or completely absent. Furthermore, the attachment of myofibrils to the junctional membrane was often aberrant or completely disrupted. These results suggest that in active muscle IFs play an important role in the organization and/or stabilization of myofibril-membrane attachment sites.

Animals↗

PubMed1995

Graph-theoretic approach to RNA modeling using comparative data.

We have examined the utility of a graph-theoretic algorithm for building comparative RNA models. The method uses a maximum weighted matching algorithm to find the optimal set of basepairs given the mutual information for all pairs of alignment positions. In all cases examined, the technique generated models similar to those based on conventional comparative analysis. Any set of pairwise interactions can be suggested including pseudoknots. Here we describe the details of the method and demonstrate its implementation on tRNA where many secondary and tertiary base-pairs are accurately predicted. We also examine the usefulness of the method for the identification of shared structural features in families of RNAs isolated by artificial selection methods such as SELEX.

Algorithms↗

PubMed1994

Desmin organization during the differentiation of the dorsal myotome in Xenopus laevis.

The reorganization of desmin-type intermediate filaments during muscle differentiation has been studied primarily in cultured cell systems. Here we describe the process of desmin reorganization during the differentiation of the dorsal myotomal muscle of the clawed frog Xenopus laevis. This muscle differs from those described previously primarily in that the desmin system forms de novo, i.e., without the presence of a pre-existing vimentin filament system. The most striking observation is that prior to myotomal segmentation and rotation desmin is concentrated at the medial and lateral tips of the myocytes. It remains concentrated in these regions following somite rotation and is located primarily to the intersomite junctions as late as the stage 33-35 tadpole. As the muscle matures (stage 30 and later) desmin becomes increasingly associated with the sarcolemma and with the Z-discs. The concentration of desmin at the nascent intersomite junction suggests that desmin is involved in coupling somites to one another in the early Xenopus embryo.

Animals↗

PubMed1994

Differential organization of desmin and vimentin in muscle is due to differences in their head domains.

In most myogenic systems, synthesis of the intermediate filament (IF) protein vimentin precedes the synthesis of the muscle-specific IF protein desmin. In the dorsal myotome of the Xenopus embryo, however, there is no preexisting vimentin filament system and desmin's initial organization is quite different from that seen in vimentin-containing myocytes (Cary and Klymkowsky, 1994. Differentiation. In press.). To determine whether the organization of IFs in the Xenopus myotome reflects features unique to Xenopus or is due to specific properties of desmin, we used the injection of plasmid DNA to drive the synthesis of vimentin or desmin in myotomal cells. At low levels of accumulation, exogenous vimentin and desmin both enter into the endogenous desmin system of the myotomal cell. At higher levels exogenous vimentin forms longitudinal IF systems similar to those seen in vimentin-expressing myogenic systems and massive IF bundles. Exogenous desmin, on the other hand, formed a reticular IF meshwork and non-filamentous aggregates. In embryonic epithelial cells, both vimentin and desmin formed extended IF networks. Vimentin and desmin differ most dramatically in their NH2-terminal "head" regions. To determine whether the head region was responsible for the differences in the behavior of these two proteins, we constructed plasmids encoding chimeric proteins in which the head of one was attached to the body of the other. In muscle, the vimentin head-desmin body (VDD) polypeptide formed longitudinal IFs and massive IF bundles like vimentin. The desmin head-vimentin body (DVV) polypeptide, on the other hand, formed IF meshworks and non-filamentous structures like desmin. In embryonic epithelial cells DVV formed a discrete filament network while VDD did not. Based on the behavior of these chimeric proteins, we conclude that the head domains of vimentin and desmin are structurally distinct and not interchangeable, and that the head domain of desmin is largely responsible for desmin's muscle-specific behaviors.

Amino Acid Sequence↗

PubMed1994

Vimentin's tail interacts with actin-containing structures in vivo.

The tail domain of the intermediate filament (IF) protein vimentin is unnecessary for IF assembly in vitro. To study the role of vimentin's tail in vivo, we constructed a plasmid that directs the synthesis of a 'myc-tagged' version of the Xenopus vimentin-1 tail domain in bacteria. This polypeptide, mycVimTail, was purified to near homogeneity and injected into cultured Xenopus A6 cells. In these cells the tail polypeptide co-localized with actin even in the presence of cytochalasin. Two myc-tagged control polypeptides argue for the specificity of this interaction. First, a similarly myc-tagged lamin tail domain localizes to the nucleus, indicating that the presence of the myc tag did not itself confer the ability to co-localize with actin (Hennekes and Nigg (1994) J. Cell Sci. 107, 1019-1029). Second, a myc-tagged polypeptide with a molecular mass and net charge at physiological pH (i.e. -4) similar to that of the mycVimTail polypeptide, failed to show any tendency to associate with actin-containing structures, indicating that the interaction between mycVimTail and actin-containing structures was not due to a simple ionic association. Franke (1987; Cell Biol. Int. Rep. 11, 831) noted a similarity in the primary sequence between the tail of the type I keratin DG81A and vimentin. To test whether the DG81A tail interacted with actin-containing structures, we constructed and purified myc-tagged DG81A tail polypeptides. Unexpectedly, these keratin tail polypeptides were largely insoluble under physiological conditions and formed aggregates at the site of injection. While this insolubility made it difficult to determine if they associated with actin-containing structures, it does provide direct evidence that the tails of vimentin and DG81A differ dramatically in their physical properties. Our data suggest that vimentin's tail domain has a highly extended structure, binds to actin-containing structures and may mediate the interaction between vimentin filaments and microfilaments involved in the control of vimentin filament organization (Hollenbeck et al. (1989) J. Cell Sci. 92, 621; Tint et al. (1991) J. Cell Sci. 98, 375).

Actins↗

PubMed1992

Host cell factors controlling vimentin organization in the Xenopus oocyte.

To study vimentin filament organization in vivo we injected Xenopus oocytes, which have no significant vimentin system of their own, with in vitro-synthesized RNAs encoding Xenopus vimentins. Exogenous vimentins were localized primarily to the cytoplasmic surface of the nucleus and to the subplasma membrane "cortex." In the cortex of the animal hemisphere, wild-type vimentin forms punctate structures and short filaments. In contrast, long anastomosing vimentin filaments are formed in the vegetal hemisphere cortex. This asymmetry in the organization of exogenous vimentin is similar to that of the endogenous keratin system (Klymkowsky, M. W., L. A. Maynell, and A. G. Polson. 1987. Development (Camb.). 100:543-557), which suggests that the same cellular factors are responsible for both. Before germinal vesicle breakdown, in the initial stage of oocyte maturation, large vimentin and keratin filament bundles appear in the animal hemisphere. As maturation proceeds, keratin filaments fragment into soluble oligomers (Klymkowsky, M. W., L. A. Maynell, and C. Nislow. 1991. J. Cell Biol. 114:787-797), while vimentin filaments remain intact and vimentin is hyperphosphorylated. To examine the role of MPF kinase in the M-phase reorganization of vimentin we deleted the conserved proline of vimentin's single MPF-kinase site; this mutation had no apparent effect on the prophase or M-phase behavior of vimentin. In contrast, deletion of amino acids 19-68 or 18-61 of the NH2-terminal "head" domain produced proteins that formed extended filaments in the animal hemisphere of the prophase oocyte. We suggest that the animal hemisphere cortex of the prophase oocyte contains a factor that actively suppresses the formation of extended vimentin filaments through a direct interaction with vimentin's head domain. During maturation this "suppressor of extended filaments" appears to be inactivated, leading to the formation of an extended vimentin filament system.

Amino Acid Sequence↗