Septin C-Terminal Domain Interactions: Implications for Filament Stability and Assembly

Septins form a conserved family of filament forming GTP binding proteins found in a wide range of eukaryotic cells. They share a common structural architecture consisting of an N-terminal domain, a central GTP binding domain and a C-terminal domain, which is often predicted to adopt a coiled-coil conformation, at least in part. The crystal structure of the human SEPT2/SEPT6/SEPT7 heterocomplex has revealed the importance of the GTP binding domain in filament formation, but surprisingly no electron density was observed for the C-terminal domains and their function remains obscure. The dearth of structural information concerning the C-terminal region has motivated the present study in which the putative C-terminal domains of human SEPT2, SEPT6 and SEPT7 were expressed in E. coli and purified to homogeneity. The thermal stability and secondary structure content of the domains were studied by circular dichroism spectroscopy, and homo- and hetero-interactions were investigated by size exclusion chromatography, chemical cross-linking, analytical ultracentrifugation and surface plasmon resonance. Our results show that SEPT6-C and SEPT7-C are able to form both homo- and heterodimers with a high alpha-helical content in solution. The heterodimer is elongated and considerably more stable than the homodimers, with a K (D) of 15.8 nM. On the other hand, the homodimer SEPT2-C has a much lower affinity, with a K (D) of 4 mu M, and a moderate alpha-helical content. Our findings present the first direct experimental evidence toward better understanding the biophysical properties and coiled-coil pairings of such domains and their potential role in filament assembly and stability.

Spermatophoric reaction reappraised: Novel insights into the functioning of the loliginid spermatophore based on Doryteuthis plei (Mollusca: Cephalopoda)

During copulation, spermatophores produced by male coleoid cephalopods undergo the spermatophoric reaction, a complex process of evagination that culminates in the attachment of the spermatangium (everted spermatophore containing the sperm mass) on the female's body. To better understand this complicated phenomenon, the present study investigated the functional morphology of the spermatophore of the squid Doryteuthis plei applying in vitro analysis of the reaction, as well as light and electron microscopy investigation of spermatangia obtained either in vitro, or naturally attached on females. Hitherto unnoticed functional features of the loliginid spermatophore require a reappraisal of some important processes involved in the spermatophoric reaction. The most striking findings concern the attachment mechanism, which is not carried out solely by cement adhesive material, as previously believed, but rather by an autonomous, complex process performed by multiple structures during the spermatophoric reaction. During evagination, the ejaculatory apparatus provides anchorage on the targeted tissue, presumably due to the minute stellate particles present in the exposed spiral filament. Consequently, the ejaculatory apparatus maintains the attachment of the tip of the evaginating spermatophore until the cement body is extruded. Subsequently, the cement body passes through a complex structural rearrangement, which leads to the injection of both its viscid contents and pointed oral region onto the targeted tissue. The inner membrane at the oral region of the cement body contains numerous stellate particles attached at its inner side; eversion of this membrane exposes these sharp structures, which presumably adhere to the tissue and augment attachment. Several naturally attached spermatangia were found with their bases implanted at the deposition sites, and the possible mechanisms of perforation are discussed based on present evidence. The function of the complex squid spermatophore and its spermatophoric reaction is revisited in light of these findings. J. Morphol. 2012. (C) 2011 Wiley Periodicals, Inc.

Unraveling the structure of squids' spermatophores: a combined approach based on Doryteuthis plei (Blainville, 1823) (Cephalopoda: Loliginidae)

Marian, J.E.A.R. and Domaneschi, O. 2012. Unraveling the structure of squids spermatophores: a combined approach based on Doryteuthis plei (Blainville, 1823) (Cephalopoda: Loliginidae). Acta Zoologica (Stockholm) 93: 281307. Male coleoid cephalopods produce elaborate spermatophores, which function autonomously outside the male body during copulation, undergoing a complicated process of evagination. In order to contribute to the understanding of this unique structure, this study investigated the morphology of the spermatophore of Doryteuthis plei applying several microscopy techniques. A hitherto unreported, much more complex structural arrangement was revealed for the loliginid spermatophore, the most striking findings being: (1) the complex, layered structure of the middle membrane, which bears an additional, chemically distinct segment surrounding part of the cement body; (2) the presence of a space between the inner tunic and middle membrane filled with a fine reticulated material; (3) the presence of stellate particles not only embedded in the spiral filament, but also closely applied to the inner membrane at the level of the cement body; (4) the presence of a pre-oral chamber in the cap region; and (5) the complex organization of the cement body, formed by two distinct layers encompassing contents of different chemical and textural properties. Careful literature reassessment suggests several of these features are common to loliginids, and to some extent to other squids. Their possible functional implications are discussed in light of our knowledge of the spermatophoric reaction mechanics.

A model to explain spermatophore implantation in cephalopods (Mollusca: Cephalopoda) and a discussion on its evolutionary origins and significance

Male coleoid cephalopods produce spermatophores that can attach autonomously on the female's body during a complex process of evagination called the spermatophoric reaction, during which the ejaculatory apparatus and spiral filament of the spermatophore are everted and exposed to the external milieu. In some deepwater cephalopods, the reaction leads to the intradermal implantation of the spermatophore, a hitherto enigmatic phenomenon. The present study builds upon several lines of evidence to propose that spermatophore implantation is probably achieved through the combination of (1) an evaginating-tube mechanism performed by the everting ejaculatory apparatus and (2) the anchorage provided by the spiral filament's stellate particles. The proposed theoretical model assumes that, as it is exposed to the external milieu, each whorl of the spiral filament anchors to the surrounding tissue by means of its sharp stellate particles. As the ejaculatory apparatus tip continues evaginating, it grows in diameter and stretches lengthwise, enlarging the diameter of the whorl and propelling it, consequently tearing and pushing the anchored tissue outward and backward, and opening space for the next whorl to attach. After the ejaculatory apparatus has been everted and has perforated tissue, the cement body is extruded, possibly aiding in final attachment, and the sperm mass comes to lie inside the female tissue, encompassed by the everted ejaculatory apparatus tube. It is proposed that this unique, efficient spermatophore attachment mechanism possibly evolved in intimate relationship with the adoption of an active mode of life by coleoids. The possible roles of predation pressure and sperm competition in the evolution of this mechanism are also discussed. (c) 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105, 711726.

A non-cross-bridge, static tension is present in permeabilized skeletal muscle fibers after active force inhibition or actin extraction

Cornachione AS, Rassier DE. A non-cross-bridge, static tension is present in permeabilized skeletal muscle fibers after active force inhibition or actin extraction. Am J Physiol Cell Physiol 302: C566-C574, 2012. First published November 16, 2011; doi: 10.1152/ajpcell.00355.2011.-When activated muscle fibers are stretched, there is a long-lasting increase in the force. This phenomenon, referred to as "residual force enhancement," has characteristics similar to those of the " static tension," a long-lasting increase in force observed when muscles are stretched in the presence of Ca2+ but in the absence of myosin-actin interaction. Independent studies have suggested that these two phenomena have a common mechanism and are caused either by 1) a Ca2+-induced stiffening of titin or by 2) promoting titin binding to actin. In this study, we performed two sets of experiments in which activated fibers (pCa(2+) 4.5) treated with the myosin inhibitor blebbistatin were stretched from 2.7 to 2.8 mu m at a speed of 40 L-o/s, first, after partial extraction of TnC, which inhibits myosin-actin interactions, or, second, after treatment with gelsolin, which leads to the depletion of thin (actin) filaments. We observed that the static tension, directly related with the residual force enhancement, was not changed after treatments that inhibit myosin-actin interactions or that deplete fibers from troponin C and actin filaments. The results suggest that the residual force enhancement is caused by a stiffening of titin upon muscle activation but not with titin binding to actin. This finding indicates the existence of a Ca2+-regulated, titin-based stiffness in skeletal muscles.