Value of models for membrane budding

The budding of membranes and curvature generation is common to many forms of trafficking in cells. Clathrin-mediated endocytosis, as a prototypical example of trafficking, has been studied in great detail using a variety of experimental systems and methods. Recently, advances in experimental methods have led to great strides in insights on the molecular mechanisms and the spatiotemporal dynamics of the protein machinery associated with membrane curvature generation. These advances have been ably supported by computational models, which have given us insights into the underlying mechanical principles of clathrin-mediated endocytosis. On the other hand, targeted experimental perturbation of membranes has lagged behind that of proteins in cells. In this area, modeling is especially critical to interpret experimental measurements in a mechanistic context. Here, we discuss the contributions made by these models to our understanding of endocytosis and identify opportunities to strengthen the connections between models and experiments.     

Plasmid instability in an industrial strain ofBacillus subtilis grown in chemostat culture

Summary A pUB110-derived plasmid/Bacillus subtilis host combination was segregationally unstable when grown in chemostat culture with complex or minimal medium and under starch, glucose or magnesium limitation. The kinetics of plasmid loss were described in terms of the difference in growth rates between plasmid-containing and plasmid-free cells (d) and the rate at which plasmid-free cells were generated from plasmid-containing cells (R). Loss of plasmid-containing cells from the population was d dominated. Changes in medium composition and the nature of growth limitation caused variations in both d and R. The plasmid was most stable in glucose-limited chemostat cultures with minimal medium and least stable under starch limitation with complex complex medium. R and d were smaller for cultures in complex media than those in minimal media. Limitation by starch induced expression of the plasmid-encoded HT amylase gene and was associated with increased values of R and d. Magnesium limitation in minimal medium caused a significant increase in d and a decrease in R.Abbreviations Cm chloramphenicol - Kan kanamycin - Cm^r cells resistant to chloramphenicol (5 mg L^–1) - Kan^r cells resistant to kanamycin (5 mg L^–1) - Cm^sKan^s cells sensitive to chloramphenicol and kanamycin     

Beyond the GTP-cap: Elucidating the molecular mechanisms of microtubule catastrophe

Almost 40 years since the discovery of microtubule dynamic instability, the molecular mechanisms underlying microtubule dynamics remain an area of intense research interest. The “standard model” of microtubule dynamics implicates a “cap” of GTP-bound tubulin dimers at the growing microtubule end as the main determinant of microtubule stability. Loss of the GTP-cap leads to microtubule “catastrophe,” a switch-like transition from microtubule growth to shrinkage. However, recent studies, using biochemical in vitro reconstitution, cryo-EM, and computational modeling approaches, challenge the simple GTP-cap model. Instead, a new perspective on the mechanisms of microtubule dynamics is emerging. In this view, highly dynamic transitions between different structural conformations of the growing microtubule end – which may or may not be directly linked to the nucleotide content at the microtubule end – ultimately drive microtubule catastrophe.     

Effects of the egg incubation environment on turtle carapace development

Developing organisms are often exposed to fluctuating environments that destabilize tissue-scale processes and induce abnormal phenotypes. This might be common in species that lay eggs in the external environment and with little parental care, such as many reptiles. In turtles, morphological development has provided striking examples of abnormal phenotypic patterns, though the influence of the environment remains unclear. To this end, we compared fluctuating asymmetry, as a proxy for developmental instability, in turtle hatchlings incubated in controlled laboratory and unstable natural conditions. Wild and laboratory hatchlings featured similar proportions of supernumerary scales (scutes) on the dorsal shell (carapace). Such abnormal scutes likely elevated shape asymmetry, which was highest in natural nests. Moreover, we tested the hypothesis that hot and dry environments cause abnormal scute formation by subjecting eggs to a range of hydric and thermal laboratory incubation regimes. Shape asymmetry was similar in hatchlings incubated at five constant temperatures (26–30°C). A hot (30°C) and severely Dry substrate yielded smaller hatchlings but scutes were not overtly affected. Our study suggests that changing nest environments contribute to fluctuating asymmetry in egg-laying reptiles, while clarifying the conditions at which turtle shell development remains buffered from the external environment.     

Proteinases and the instability of isocitrate lyase in extracts of developing flax seedlings

In extracts of flax seedlings 4 days after imbibition, isocitrate lyase activity is unstable in comparison to that in extracts from 2.5-day seedlings or to malate syntheses analysed at several stages of development. This instability in extracts of 4-day seedlings is especially pronounced when a large number of seedlings is homogenized per unit volume of Tris-Mg²⁺-EDTA-dithioerythritol buffer. However, isocitrate lyase can be stabilized when the resultant homogenate is diluted soon after seedling breakage. The pronounced instability in more concentrated extracts is not due to inadequate buffering by the homogenization medium, nor can it be due to polyphenols because added polyvinylpyrrolidone has no effect. Mixing of a heated supernatant from concentrated extract with dilute unheated extract yields the units of stable isocitrate lyase expected in the dilute extract, ruling out stoichiometric inactivation by a heat-stable component. The pronounced instability is attributed to the action of proteinases. A theoretical model assuming a decay process that is first order in isocitrate lyase and first-order in one or more proteinases is in reasonable agreement with the results. Malate synthase and NADP⁺-isocitrate dehydrogenase are much more stable in concentrated extracts prepared from 4-day flax seedlings. Isocitrate lyase is stable in concentrated extracts of 5-day watermelon seedlings, which is a developmental stage analogous to that for 4-day flax seedlings.     

Genome instability and embryonic developmental defects in RMI1 deficient mice

RMI1 forms an evolutionarily conserved complex with BLM/TOP3α/RMI2 (BTR complex) to prevent and resolve aberrant recombination products, thereby promoting genome stability. Most of our knowledge about RMI1 function has been obtained from biochemical studies in vitro. In contrast, the role of RMI1 in vivo remains unclear. Previous attempts to generate an Rmi1 knockout mouse line resulted in pre-implantation embryonic lethality, precluding the use of mouse embryonic fibroblasts (MEFs) and embryonic morphology to assess the role of RMI1 in vivo. Here, we report the generation of an Rmi1 deficient mouse line (hy/hy) that develops until 9.5 days post coitum (dpc) with marked defects in development. MEFs derived from Rmi1^hy/hy are characterized by severely impaired cell proliferation, frequently having elevated DNA content, high numbers of micronuclei and an elevated percentage of partial condensed chromosomes. Our results demonstrate the importance of RMI1 in maintaining genome integrity and normal embryonic development.