Worawit Suphamungmee

The flexibility of two tropomyosin mutants, D175N and E180G, that cause hypertrophic cardiomyopathy

Point mutations targeting muscle thin filament proteins are the cause of a number of cardiomyopathies. In many cases, biological effects of the mutations are well-documented, whereas their structural and mechanical impact on filament assembly and regulatory function is lacking. In order to elucidate molecular defects leading to cardiac dysfunction, we have examined the structural mechanics of two tropomyosin mutants, E180G and D175N, which are associated with hypertrophic cardiomyopathy (HCM). Tropomyosin is an α-helical coiled-coil dimer which polymerizes end-to-end to create an elongated superhelix that wraps around F-actin filaments of muscle and non-muscle cells, thus modulating the binding of other actin-binding proteins. Here, we study how flexibility changes in the E180G and D175N mutants might affect tropomyosin binding and regulatory motion on F-actin. Electron microscopy and Molecular Dynamics simulations show that E180G and D175N mutations cause an increase in bending flexibility of tropomyosin both locally and globally. This excess flexibility is likely to increase accessibility of the myosin-binding sites on F-actin, thus destabilizing the low-Ca(2+) relaxed-state of cardiac muscle. The resulting imbalance in the on-off switching mechanism of the mutants will shift the regulatory equilibrium towards Ca(2+)-activation of cardiac muscle, as is observed in affected muscle, accompanied by enhanced systolic activity, diastolic dysfunction, and cardiac compensations associated with HCM and heart failure.

FlnA binding to PACSIN2 F-BAR domain regulates membrane tubulation in megakaryocytes and platelets

Bin-Amphiphysin-Rvs (BAR) and Fes-CIP4 homology BAR (F-BAR) proteins generate tubular membrane invaginations reminiscent of the megakaryocyte (MK) demarcation membrane system (DMS), which provides membranes necessary for future platelets. The F-BAR protein PACSIN2 is one of the most abundant BAR/F-BAR proteins in platelets and the only one reported to interact with the cytoskeletal and scaffold protein filamin A (FlnA), an essential regulator of platelet formation and function. The FlnA-PACSIN2 interaction was therefore investigated in MKs and platelets. PACSIN2 associated with FlnA in human platelets. The interaction required FlnA immunoglobulin-like repeat 20 and the tip of PACSIN2 F-BAR domain and enhanced PACSIN2 F-BAR domain membrane tubulation in vitro. Most human and wild-type mouse platelets had 1 to 2 distinct PACSIN2 foci associated with cell membrane GPIbα, whereas Flna-null platelets had 0 to 4 or more foci. Endogenous PACSIN2 and transfected enhanced green fluorescent protein-PACSIN2 were concentrated in midstage wild-type mouse MKs in a well-defined invagination of the plasma membrane reminiscent of the initiating DMS and dispersed in the absence of FlnA binding. The DMS appeared less well defined, and platelet territories were not readily visualized in Flna-null MKs. We conclude that the FlnA-PACSIN2 interaction regulates membrane tubulation in MKs and platelets and likely contributes to DMS formation.

Cryotomography of Budding Influenza A Virus Reveals Filaments with Diverse Morphologies that Mostly Do Not Bear a Genome at Their Distal End

Influenza viruses exhibit striking variations in particle morphology between strains. Clinical isolates of influenza A virus have been shown to produce long filamentous particles while laboratory-adapted strains are predominantly spherical. However, the role of the filamentous phenotype in the influenza virus infectious cycle remains undetermined. We used cryo-electron tomography to conduct the first three-dimensional study of filamentous virus ultrastructure in particles budding from infected cells. Filaments were often longer than 10 microns and sometimes had bulbous heads at their leading ends, some of which contained tubules we attribute to M1 while none had recognisable ribonucleoprotein (RNP) and hence genome segments. Long filaments that did not have bulbs were infrequently seen to bear an ordered complement of RNPs at their distal ends. Imaging of purified virus also revealed diverse filament morphologies; short rods (bacilliform virions) and longer filaments. Bacilliform virions contained an ordered complement of RNPs while longer filamentous particles were narrower and mostly appeared to lack this feature, but often contained fibrillar material along their entire length. The important ultrastructural differences between these diverse classes of particles raise the possibility of distinct morphogenetic pathways and functions during the infectious process.

Regulation of dynamin oligomerization in cells: the role of dynamin-actin interactions and its GTPase activity.

Dynamin is a 96‐kDa protein that has multiple oligomerization states that influence its GTPase activity. A number of different dynamin effectors, including lipids, actin filaments, and SH3‐domain‐containing proteins, have been implicated in the regulation of dynamin oligomerization, though their roles in influencing dynamin oligomerization have been studied predominantly in vitro using recombinant proteins. Here, we identify higher order dynamin oligomers such as rings and helices in vitro and in live cells using fluorescence lifetime imaging microscopy (FLIM). FLIM detected GTP‐ and actin‐dependent dynamin oligomerization at distinct cellular sites, including the cell membrane and transition zones where cortical actin transitions into stress fibers. Our study identifies a major role for direct dynamin–actin interactions and dynamins GTPase activity in the regulation of dynamin oligomerization in cells.

Polymorphism in tropomyosin structure and function

Tropomyosins (Tm) in humans are expressed from four distinct genes and by alternate splicing >40 different Tm polypeptide chains can be made. The functional Tm unit is a dimer of two parallel polypeptide chains and these can be assembled from identical (homodimer) or different (heterodimer) polypeptide chains provided both chains are of the same length. Since most cells express multiple isoforms of Tm, the number of different homo and heterodimers that can be assembled becomes very large. We review the mechanism of dimer assembly and how preferential assembly of some heterodimers is driven by thermodynamic stability. We examine how in vitro studies can reveal functional differences between Tm homo and heterodimers (stability, actin affinity, flexibility) and the implication for how there could be selection of Tm isomers in the assembly on to an actin filament. The role of Tm heterodimers becomes more complex when mutations in Tm are considered, such as those associated with cardiomyopathies, since mutations can appear in only one of the chains.

Ultrastructure, Composition, and Possible Roles of the Egg Coats in Haliotis asinina

ABSTRACT Spawned eggs of a tropical abalone Haliotis asinina have 2 protective barriers: the egg jelly coat and the vitelline envelope. At the electron microscopic level, the egg jelly is composed of a network of large fibers (40–50 nm thick) cross-linked by smaller fibers (15–20 nm thick), whereas the vitelline envelope is a thin, tough sheet containing pores that might be channels for sperm contact and entry. Electrophoretically, the egg jelly contains 2 major glycoproteins at 107 kDa and 178 kDa, whereas the vitelline envelope contains a broad spectrum of protein bands ranging from 15–200 kDa, which also includes the corresponding egg jelly protein bands. Glycoproteins of egg jelly and vitelline envelope exhibit strong cross-reactivities, and they appear in late oocytes (Oc4, Oc5). Glucose is the major sugar composition of both egg jelly and vitelline envelope glycoproteins, whereas minor proportions of arabinose, fructose, galactose, and fucose are present in both the egg jelly and vitelline envelope. Our findings suggest that a sperm acrosome reaction could be induced by isolated vitelline envelope glycoproteins, whereas acceleration of sperm motility could be stimulated by egg jelly glycoproteins.

Electron Microscopy and 3D Reconstruction Reveals Filamin Ig Domain Binding to F-Actin

Filamin A (FLNa) is an actin-binding protein that cross-links F-actin into networks of orthogonally branched filaments. FLNa also directs the networks to integrins while responding to mechanochemical signaling pathways. Flexible, 160-nm-long FLNa molecules are tail-to-tail dimers, each subunit of which contains an N-terminal calponin homology (CH)/actin-binding domain connected by a series of 24 immunoglobulin (Ig) repeats to a dimerization site at their C-terminal end. Whereas the contribution of the CH domains to F-actin affinity is weak (apparent K(a)~10(5)), the binding of the intact protein to F-actin is strong (apparent K(a)~10(8)), suggesting involvement of additional parts of the molecule in this association. Indeed, previous results indicate that Ig repeats along FLNa contribute significantly to the strength of the actin filament interaction. In the current study, we used electron microscopy and three-dimensional reconstruction to elucidate the structural basis of the Ig repeat-F-actin binding. We find that FLNa density is clearly delineated in reconstructions of F-actin complexed either with a four-Ig-repeat segment of FLNa containing Ig repeat 10 or with immunoglobulin-like filamin A repeat (IgFLNa)10 alone. The mass attributable to IgFLNa10 lies peripherally along the actin helix over the N-terminus of actin subdomain 1. The IgFLNa10 interaction appears to be specific, since no other individual Ig repeat or fragment of the FLNa molecule examined, besides ones with IgFLNa10 or CH domains, decorated F-actin filaments or were detected in reconstructions. We conclude that the combined interactions of CH domains and the IgFLNa10 repeat provide the binding strength of the whole FLNa molecule and propose a model for the association of IgFLNa10 on actin filaments.

Starvation Promotes Autophagy-Associated Maturation of the Ovary in the Giant Freshwater Prawn, Macrobrachium rosenbergii

Limitation of food availability (starvation) is known to influence the reproductive ability of animals. Autophagy is a lysosomal driven degradation process that protects the cell under metabolic stress conditions, such as during nutrient shortage. Whether, and how starvation-induced autophagy impacts on the maturation and function of reproductive organs in animals are still open questions. In this study, we have investigated the effects of starvation on histological and cellular changes that may be associated with autophagy in the ovary of the giant freshwater prawn, Macrobachium rosenbergii. To this end, the female prawns were daily fed (controls) or unfed (starvation condition) for up to 12 days, and the ovary tissue was analyzed at different time-points. Starvation triggered ovarian maturation, and concomitantly increased the expression of autophagy markers in vitellogenic oocytes. The immunoreactivities for autophagy markers, including Beclin1, LC3-II, and Lamp1, were enhanced in the late oocytes within the mature ovaries, especially at the vitellogenic stages. These markers co-localized with vitellin in the yolk granules within the oocytes, suggesting that autophagy induced by starvation could drive vitellin utilization, thus promoting ovarian maturation.

Proportion of Sperm and Eggs for Maximal in vitro Fertilization in Haliotis asinina and the Chronology of Early Development

ABSTRACT To obtain the highest yield during in vitro fertilization of tropical abalone Haliotis asinina, optimal proportion of the gametes, the timing of sperm-egg interaction, and subsequent development were investigated. The highest yield of fertilization (75%) with fewest abnormal eggs was obtained when incubating eggs and sperm at the ratio of 1:100 in seawater with a salinity of 27.5 ppt, a pH of 7.8, and a temperature range from 27–29°C. After incubation, sperm swim through the eggjelly coat and become bound to the vitelline envelope within 30 sec, followed by an acrosomal reaction at 1 min. The fertilized egg extrudes the first and second polar bodies at 8–10 min, and then the zygote begins cleavage at 15–20 min. This is followed by the second cleavage, and development through the stages of blastula, gastrula, trochophore, veliger, and early creeping larvae, which were completed within 3 days. Noticeably, occurrence of egg jelly condensation after penetration of the first sperm would not allow other sperm bind to the egg jelly and to penetrate through its vitelline envelope. This event is thought to be a weak blocking against polyspermy, because the classic cortical reaction initiated by cortical granule exocytosis could not be observed in this species.

Distribution and dynamic expression of serotonin and dopamine in the nervous system and ovary of Holothuria scabra during ovarian maturation

In the present study, the distribution and dynamic expression of serotonin and dopamine in the nervous system and ovary of the sea cucumber, Holothuria scabra, during different ovarian stages were investigated. We found that serotonin-immunoreactivity was more intense in the neurons and neuropils of the outer ectoneural part, the inner hyponeural part, and the wall of hyponeural canal of radial nerve cord during the mature stages of ovarian cycle, whereas dopamine-immunoreactivity was detected at a higher intensity in these tissues during the early stages. Both neurotransmitters were detected in the ectoneural part of the nerve ring. In the ovary, serotonin intensity was more intense in the cytoplasm of late oocytes, while dopamine-immunoreactivity was more intense in the early stages. The changes in the levels serotonin in the radial nerve cord and oocytes are incremental towards the late stages of ovarian maturation. In contrast, dopamine levels in the nervous tissues and oocytes were more intense in early stages and became decremental towards the late stages. These findings suggest that serotonin and dopamine may have opposing effects on ovarian development in this sea cucumber species.