Visual acuity in larval zebrafish: behavior and histology

Background

Mating plugs that males place onto the female genital tract are generally assumed to prevent remating with other males. Mating plugs are usually explained as a consequence of male-male competition in multiply mating species. Here, we investigated whether mating plugs also have collateral effects on female fitness. These effects are negative when plugging reduces female mating rate below an optimum. However, plugging may also be positive when plugging prevents excessive forced mating and keeps mating rate closer to a females' optimum. Here, we studied these consequences in the gonochoristic nematode Caenorhabditis remanei. We employed a new CO₂-sedation technique to interrupt matings before or after the production of a plug. We then measured mating rate, attractiveness and offspring number.

Results

The presence of a mating plug did not affect mating rate or attractiveness to roving males. Instead, females with mating plugs produced more offspring than females without copulatory plugs.

Conclusions

Our experiment suggests that plugging might have evolved under male-male competition but represents a poor protection against competing males in our experiment. Even if plugging does not reduce mating rate, our results indicate that females may benefit from being plugged in a different sense than remating prevention.     

Analysis of the interacting partners of the neuronal calcium-binding proteins L-CaBP1, hippocalcin, NCS-1 and neurocalcin delta

Intracellular Ca2+ signals are transduced by the binding of Ca2+ to sensor proteins, which subsequently modify the activity of their target proteins. Identification of these target proteins is, therefore, important for an understanding of cellular signalling processes. We have investigated the binding partners of four EF-hand Ca2+-binding proteins. Three proteins of the neuronal calcium sensor (NCS) family, hippocalcin, NCS-1 and neurocalcin delta were prepared as N-terminally tagged GST fusion proteins, and the less closely related protein L-CaBP1 was prepared in both N- and C-terminally tagged forms, the latter requiring generation of a new vector. Immobilised fusion proteins were used to purify binding partners from bovine brain cytosol and membrane extracts in the presence of 1 microM free Ca2+. Bound proteins were eluted with Ca2+-free and high-salt buffers and eluted proteins were identified by MALDI-MS and Western blotting. New protein targets detected included ARF1, Ca2+-dependent activator protein for secretion 1, cyclic nucleotide 3',5'-phosphodiesterase, the vacuolar ATPase, AP1 and AP2 complexes and the type I TGF-beta receptor. While certain of these interactions occurred with more than one of the Ca2+-binding proteins, others were found to be specific targets for particular Ca2+ sensors, and many of these did not overlap with known calmodulin-binding proteins. These findings provide new clues to the functional roles of the neuronal calcium sensor proteins.     

Interpretation of the properties of chromatin extracts from mammalian nuclei

Chromatin from standard preparations of nuclei stabilized by magnesium ions at 0-4 degrees C was degraded during the nuclear isolation, and newly-synthesized chromatin was degraded slightly more slowly than the ;older' chromatin. The significance of this observation, and its relation to the interpretation of the properties of nucleoprotein extracts is discussed.     

The mechanisms of pyknosis: hypercondensation and death

Intense nuclear condensation with intense refractivity (pyknosis) is the ubiquitous terminus of all apoptosis and some necrosis of vertebrate cells, but its structural basis is unknown. Intense condensations were induced in a model system, the avian erythrocyte, and three different molecular processes distinguished from each other. Two of the hypercondensations, nucleolytic pyknosis, as in mammalian apoptosis, and anucleolytic pyknosis, as in necrosis, appear to be energetically spontaneous and appear to have a conformational basis with the third hypercondensation being a trans-nuclear membrane osmotic pressure compression effect. Nucleolytic pyknosis as per apoptosis was not intrinsic in this system and required exogenous nuclease. The pure anucleolytic pyknosis supported by this system was not induced by the apoptopic induction agents, staurosporine or antitopoisomerases (I and II), indicating a simple but unusual signaling pathway for anucleolytic pyknosis. Molecular weight determinations of the H5, H3, H4, H2a, and H2b, with final errors of +/-1 Da or less, seem to eliminate histone modifications as the basis of anucleolytic pyknosis. The molecular basis of pyknosis is proposed to be from internucleosomal rotational angle freedom that permits internucleosomal sharing of basic histone tails of adjacent nucleosomes and nucleofilaments. Much of the favorable conformational energy of pyknosis may be from the entropy increase of tail delocalization.     

Tying everything together: the multiple roles of cysteine string protein (CSP) in regulated exocytosis

In addition to the core vesicle fusion machinery, the SNARE proteins, a large number of regulatory proteins have been implicated in the process of Ca²⁺-dependent exocytosis. How these exocytotic proteins are properly targeted and how their myriad interactions are temporally and spatially coordinated is poorly understood. Cysteine string protein (CSP), a secretory vesicle membrane protein and a member of the dnaJ family of co-chaperones, may assist in performing this function. Through its interaction with the ubiquitous chaperone, Hsc70, it is thought that cysteine string protein targets chaperone complexes to the exocytotic machinery to facilitate the correct folding of polypeptides or to regulate the assembly of protein complexes. Since its discovery, there have been conflicting reports from different systems concerned with whether cysteine string protein exerts its effects on exocytosis either up- or down-stream of Ca²⁺-influx. In this review, we summarize recent experiments that associate cysteine string protein with the regulation of vesicle filling, vesicle docking, Ca²⁺-channels and the SNARE proteins themselves, hence supporting a role for cysteine string protein as a multifunctional secretory co-chaperone. In addition, we provide an update on the mammalian isoforms of cysteine string protein following the recent discovery of two novel cysteine string proteins.     

Complexin regulates the closure of the fusion pore during regulated vesicle exocytosis

Membrane fusion during exocytosis and throughout the cell is believed to involve members of the SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) family of proteins. The assembly of these proteins into a four-helix bundle may be part of the driving force for bilayer fusion. Regulated exocytosis in neurons and related cell types is specialized to be fast and Ca(2+)-dependent suggesting the involvement of other regulatory proteins specific for regulated exocytosis. Among these are the complexins, two closely related proteins that bind only to the assembled SNARE complex. We have investigated the function of complexin by analysis of single vesicle release events in adrenal chromaffin cells using carbon fiber amperometry. These cells express complexin II, and overexpression of this protein modified the kinetics of vesicle release events so that their time course was shortened. This effect depended on complexin interaction with the SNARE complex as introduction of a mutation of Arg-59, a residue that interacts with synaptobrevin in the SNARE complex, abolished its effects. The data are consistent with a function for complexin in stabilizing an intermediate of the SNARE complex to allow kiss-and-run recycling of the exocytosed vesicle.     

Localized Ca2+ uncaging reveals polarized distribution of Ca2+-sensitive Ca2+ release sites: mechanism of unidirectional Ca2+ waves

Ca2+-induced Ca2+ release (CICR) plays an important role in the generation of cytosolic Ca2+ signals in many cell types. However, it is inherently difficult to distinguish experimentally between the contributions of messenger-induced Ca2+ release and CICR. We have directly tested the CICR sensitivity of different regions of intact pancreatic acinar cells using local uncaging of caged Ca2+. In the apical region, local uncaging of Ca2+ was able to trigger a CICR wave, which propagated toward the base. CICR could not be triggered in the basal region, despite the known presence of ryanodine receptors. The triggering of CICR from the apical region was inhibited by a pharmacological block of ryanodine or inositol trisphosphate receptors, indicating that global signals require coordinated Ca2+ release. Subthreshold agonist stimulation increased the probability of triggering CICR by apical uncaging, and uncaging-induced CICR could activate long-lasting Ca2+ oscillations. However, with subthreshold stimulation, CICR could still not be initiated in the basal region. CICR is the major process responsible for global Ca2+ transients, and intracellular variations in sensitivity to CICR predetermine the activation pattern of Ca2+ waves.     

A sticker-based model for DNA computation.

We introduce a new model of molecular computation that we call the sticker model. Like many previous proposals it makes use of DNA strands as the physical substrate in which information is represented and of separation by hybridization as a central mechanism. However, unlike previous models, the stickers model has a random access memory that requires no strand extension and uses no enzymes; also (at least in theory), its materials are reusable. The paper describes computation under the stickers model and discusses possible means for physically implementing each operation. Finally, we go on to propose a specific machine architecture for implementing the stickers model as a microprocessor-controlled parallel robotic workstation. In the course of this development a number of previous general concerns about molecular computation (Smith, 1996; Hartmanis, 1995; Linial et al., 1995) are addressed. First, it is clear that general-purpose algorithms can be implemented by DNA-based computers, potentially solving a wide class of search problems. Second, we find that there are challenging problems, for which only modest volumes of DNA should suffice. Third, we demonstrate that the formation and breaking of covalent bonds is not intrinsic to DNA-based computation. Fourth, we show that a single essential biotechnology, sequence-specific separation, suffices for constructing a general-purpose molecular computer. Concerns about errors in this separation operation and means to reduce them are addressed elsewhere (Karp et al., 1995; Roweis and Winfree, 1999). Despite these encouraging theoretical advances, we emphasize that substantial engineering challenges remain at almost all stages and that the ultimate success or failure of DNA computing will certainly depend on whether these challenges can be met in laboratory investigations.