Linda Lund

Perilipin 5, a lipid droplet-associated protein, provides physical and metabolic linkage to mitochondria

Maintaining cellular lipid homeostasis is crucial to oxidative tissues, and it becomes compromised in obesity. Lipid droplets (LD) play a central role in lipid homeostasis by mediating fatty acid (FA) storage in the form of triglyceride, thereby lowering intracellular levels of lipids that mediate cellular lipotoxicity. LDs and mitochondria have interconnected functions, and anecdotal evidence suggests they physically interact. However, the mechanisms of interaction have not been identified. Perilipins are LD-scaffolding proteins and potential candidates to play a role in their interaction with mitochondria. We examined the contribution of LD perilipin composition to the physical and metabolic interactions between LD and mitochondria using multiple techniques: confocal imaging, electron microscopy (EM), and lipid storage and utilization measurements. Using neonatal cardiomyocytes, reconstituted cell culture models, and rodent heart tissues, we found that perilipin 5 (Plin5) recruits mitochondria to the LD surface through a C-terminal region. Compared with control cells, Plin5-expressing cells show decreased LD hydrolysis, decreased palmitate β-oxidation, and increased palmitate incorporation into triglycerides in basal conditions, whereas in stimulated conditions, LD hydrolysis inhibition is lifted and FA released for β-oxidation. These results suggest that Plin5 regulates oxidative LD hydrolysis and controls local FA flux to protect mitochondria against excessive exposure to FA during physiological stress.

Axonal regrowth upregulates β-actin and Jun D mRNA expression

When quiescent cells are perturbed, mRNAs encoding proteins that regulate gene transcription and the cell cycle are expressed at higher levels. Jun and Fos are examples of proteins that mediate mitogenic signals and influence differentiation. In neurons, axon interruption (axotomy) increases the content of actin, tubulin, Jun D, and c-Jun proteins in association with increases in actin mRNA levels. Jun D protein binds to gene promoter regions, and its expression has been linked to several aspects of cell differentiation. Because Jun D and β-actin messages have been described as “constitutive” in expression, we wanted to know whether these messages were responsive to axotomizing lesions of the sciatic motor nerve. We crushed the right sciatic nerve in Sprague-Dawley rats and extracted mRNA from the half spinal cord that serves each leg. At 4 days, Northern blots showed a 2.3-fold increase in β-actin mRNA and a 2.5-fold increase in Jun D mRNA in the right hemicord. In situ hybridization showed either an undiminished or increased concentration of both mRNAs in motor neurons ipsilateral to the lesion at 4 days, even though many had enlarged two-to threefold. By introducing Fluoro-Ruby at the axotomy site, we were able to show that only the axotomized neurons had enlarged. We conclude that aspects of axonal regeneration resemble the embryonic program for neuronal differentiation and are reinitiated by axotomy.

Myopathic changes in murine skeletal muscle lacking synemin.

Diseases of striated muscle linked to intermediate filament (IF) proteins are associated with defects in the organization of the contractile apparatus and its links to costameres, which connect the sarcomeres to the cell membrane. Here we study the role in skeletal muscle of synemin, a type IV IF protein, by examining mice null for synemin (synm-null). Synm-null mice have a mild skeletal muscle phenotype. Tibialis anterior (TA) muscles show a significant decrease in mean fiber diameter, a decrease in twitch and tetanic force, and an increase in susceptibility to injury caused by lengthening contractions. Organization of proteins associated with the contractile apparatus and costameres is not significantly altered in the synm-null. Elastimetry of the sarcolemma and associated contractile apparatus in extensor digitorum longus myofibers reveals a reduction in tension consistent with an increase in sarcolemmal deformability. Although fatigue after repeated isometric contractions is more marked in TA muscles of synm-null mice, the ability of the mice to run uphill on a treadmill is similar to controls. Our results suggest that synemin contributes to linkage between costameres and the contractile apparatus and that the absence of synemin results in decreased fiber size and increased sarcolemmal deformability and susceptibility to injury. Thus synemin plays a moderate but distinct role in fast twitch skeletal muscle.

INTRA-AXONAL MYOSIN AND ACTIN IN NERVE REGENERATION

A FOCUSED REVIEW of sciatic nerve regeneration in the rat model, based on research conducted by the authors, is presented. We examine structural proteins carried distally in the axon by energy-requiring motor enzymes, using protein chemistry and molecular biology techniques in combination with immunohistochemistry. Relevant findings from other laboratories are cited and discussed. The general conclusion is that relatively large amounts of actin and tubulin are required to construct a regenerating axon and that these materials mainly originate in the parent axon. The motor enzymes that carry these proteins forward as macromolecules include kinesin and dynein but probably also include myosin.

Chromodomain Helicase Binding Protein 8 (Chd8) Is a Novel A-Kinase Anchoring Protein Expressed during Rat Cardiac Development

A-kinase anchoring proteins (AKAPs) bind the regulatory subunits of protein kinase A (PKA) and localize the holoenzyme to discrete signaling microdomains in multiple subcellular compartments. Despite emerging evidence for a nuclear pool of PKA that rapidly responds to activation of the PKA signaling cascade, only a few AKAPs have been identified that localize to the nucleus. Here we show a PKA-binding domain in the amino terminus of Chd8, and demonstrate subcellular colocalization of Chd8 with RII. RII overlay and immunoprecipitation assays demonstrate binding between Chd8-S and RIIα. Binding is abrogated upon dephosphorylation of RIIα. By immunofluorescence, we identified nuclear and perinuclear pools of Chd8 in HeLa cells and rat neonatal cardiomyocytes. We also show high levels of Chd8 mRNA in RNA extracted from post-natal rat hearts. These data add Chd8 to the short list of known nuclear AKAPs, and implicate a function for Chd8 in post-natal rat cardiac development.

Synemin Isoforms Differentially Organize Cell Junctions and Desmin Filaments in Neonatal Cardiomyocytes

Intermediate filaments (IFs) in cardiomyocytes consist primarily of desmin, surround myofibrils at Z disks, and transmit forces from the contracting myofilaments to the cell surface through costameres at the sarcolemma and desmosomes at intercalated disks. Synemin is a type IV IF protein that forms filaments with desmin and also binds α‐actinin and vinculin. Here we examine the roles and expression of the α and β forms of synemin in developing rat cardiomyocytes. Quantitative PCR showed low levels of expression for both synemin mRNAs, which peaked at postnatal day 7. Synemin was concentrated at sites of cell‐cell adhesion and at Z disks in neonatal cardiomyocytes. Overexpression of the individual isoforms showed that α‐synemin preferentially localized to cell‐cell junctions, whereas P‐synemin was primarily at the level of Z disks. An siRNA targeted to both synemin isoforms reduced protein expression in cardiomyocytes by 70% and resulted in a failure of desmin to align with Z disks and disrupted cell‐cell junctions, with no effect on sarcomeric organization. Solubility assays showed that β‐synemin was soluble and interacted with sarcomeric α‐actinin by coimmunoprecipitation, while α‐synemin and desmin were insoluble. We conclude that β‐synemin mediates the association of desmin IFs with Z disks, whereas α‐synemin stabilizes junctional complexes between cardiomyocytes.—Lund, L. M., Kerr, J. P., Lupinetti, J., Zhang, Y., Russell, M. A., Bloch, R. J., Bond, M. Synemin isoforms differentially organize cell junctions and desmin filaments in neonatal cardiomyocytes. FASEB J. 26, 137–148 (2012). www.fasebj.org

Characterization of skeletal muscle in the synemin knock-out mouse

Diseases linked to intermediate filament (IF) proteins are associated with defects in the organization of the contractile apparatus of skeletal and cardiac muscle and its links to costameres, which connect the sarcomeres to the cell membrane. Synemin is a large IF protein that associates with dystrobrevin, vinculin, and talin at costameres of the cell membrane of striated muscle, as well as with α-actinin and desmin at the Z disks. Synemin can be expressed in either 210 kDa α- or 180 kDa β- alternatively spliced forms. We generated mice null for synemin by homologous recombination to study synemins function in skeletal muscle. Skeletal muscle in the knock out (syn KO) mouse does not make synemin mRNA or protein. Preliminary characterization of the syn KO mouse suggests that it has a mild skeletal muscle phenotype. The organization of costameres appears to be normal. Treadmill running uphill test results was not significantly affected when compared to controls at any age. More notably, the biomechanical p...

Myopathic Changes in Murine Skeletal Muscle Lacking Synemin

Diseases of striated muscle linked to intermediate filament (IF) proteins are associated with defects in the organization of the contractile apparatus and its links to costameres, which connect the sarcomeres to the cell membrane. Here we study the role in skeletal muscle of synemin, a type IV IF protein, by examining mice null for synemin (synm-null). Synm-null mice have a mild skeletal muscle phenotype. Tibialis anterior muscles show a significant decrease in mean fiber diameter, in twitch and tetanic tension and an increase in susceptibility to injury caused by lengthening contractions. Organization of proteins associated with the contractile apparatus and costameres is not significantly altered in the synm-null. Elastimetry of the sarcolemma and associated contractile apparatus in extensor digitorum longus myofibers reveals a reduction in stiffness consistent with an increase in sarcolemmal deformability. Although fatigue with repeated isometric contractions is greater in isolated TA muscles of synm-null mice, the ability of the mice to run uphill on a treadmill is similar to controls. Our results show that synemin contributes to linkage between costameres and the contractile apparatus, and that the absence of synemin results in decreased fiber size and increased sarcolemmal deformability and susceptibility to injury. Synemin plays a moderate but distinct role in fast twitch skeletal muscle. Supported partially by a Physiological Genomics and a CONACyT fellowship to KPGP, and by NIH to RML (R01 AR 059179), to MB (RO1 02520711), and to RJB (R01 AR 055928).