Assembly Kinetics of Vimentin Tetramers to Unit-Length Filaments: A Stopped-Flow Study

Intermediate filaments (IFs) are principal components of the cytoskeleton, a dynamic integrated system of structural proteins that provides the functional architecture of metazoan cells. They are major contributors to the elasticity of cells and tissues due to their high mechanical stability and intrinsic flexibility. The basic building block for the assembly of IFs is a rod-like, 60-nm-long tetrameric complex made from two antiparallel, half-staggered coiled coils. In low ionic strength, tetramers form stable complexes that rapidly assemble into filaments upon raising the ionic strength. The first assembly products, “frozen” by instantaneous chemical fixation and viewed by electron microscopy, are 60-nm-long “unit-length” filaments (ULFs) that apparently form by lateral in-register association of tetramers. ULFs are the active elements of IF growth, undergoing longitudinal end-to-end annealing with one another and with growing filaments. Originally, we have employed quantitative time-lapse atomic force and electron microscopy to analyze the kinetics of vimentin-filament assembly starting from a few seconds to several hours. To obtain detailed quantitative insight into the productive reactions that drive ULF formation, we now introduce a “stopped-flow” approach in combination with static light-scattering measurements. Thereby, we determine the basic rate constants for lateral assembly of tetramers to ULFs. Processing of the recorded data by a global fitting procedure enables us to describe the hierarchical steps of IF formation. Specifically, we propose that tetramers are consumed within milliseconds to yield octamers that are obligatory intermediates toward ULF formation. Although the interaction of tetramers is diffusion controlled, it is strongly driven by their geometry to mediate effective subunit targeting. Importantly, our model conclusively reflects the previously described occurrence of polymorphic ULF and mature filaments in terms of their number of tetramers per cross section.     

Combining otolith chemistry and telemetry to assess diadromous migration in pinkeye mullet, <Emphasis Type="Italic">Trachystoma petardi</Emphasis> (Actinopterygii,Mugiliformes)

Little is currently known regarding microbial community structure, and the environmental factors influencing it, within the anchialine ecosystem, defined as near-shore, land-locked water bodies with subsurface connections to the ocean and groundwater aquifer. The Hawaiian Archipelago is home to numerous anchialine habitats, with some on the islands of Maui and Hawaii harboring unique, laminated orange cyanobacterial–bacterial crusts that independently assembled in relatively young basalt fields. Here, benthic and water column bacterial and micro-eukaryotic communities from nine anchialine habitats on Oahu, Maui, and Hawaii were surveyed using high-throughput amplicon sequencing of the V6 (Bacteria-specific) and V9 (Eukarya-biased) hypervariable regions of the 16S- and 18S-rDNA genes, respectively. While benthic communities from habitats with cyanobacterial–bacterial crusts were more similar to each other than to ones lacking it on the same island, each habitat had distinct benthic and water column microbial communities. Analyses of the survey data in the context of environmental factors identified salinity, site, aquifer, and watershed as having the highest explanatory power for the observed variation in microbial diversity and community structure, with lesser drivers being annual rainfall, longitude, ammonium, and dissolved organic carbon. Our results epitomize the abiotic and biotic uniqueness characteristic of individual habitats comprising the Hawaiian anchialine ecosystem.     

Effect of fish length and nutritional condition on the fecundity of distressed Atlantic cod Gadus morhua from the Baltic Sea

The disappearance of larger individuals and the decrease in individual body condition suffered by Atlantic cod Gadus morhua in the eastern Baltic during the past two decades can be expected to affect the stock reproductive output. To investigate this, female G. morhua were collected during the spawning and pre‐spawning period in 2015?2016. The current individual potential fecundity (F_P) of eastern Baltic G. morhua was estimated and analysed in relation to total length (L_T) and indices of nutritional status such as body condition (K) and hepato‐somatic index (I_H) using generalized linear models. In addition, the current prevalence of atresia and its potential relation to K were investigated. Moreover, a calibration curve to estimate F_P from oocyte diameter, based on the autodiametric oocyte counting method, was established for the first time for eastern Baltic G. morhua and can be used for future fecundity studies on this stock. The results showed that F_P was mainly positively related to fish length, but K and I_H also contributed significantly to the variation in F_P. The model predicted that fish with K = 1·2 have a F_P 51% higher than fish of the same L_T with K = 0·8. The prevalence of fecundity regulation by atresia was 5·8%, but it was found only in fish in the pre‐spawning maturity stage and with low K. Temporal changes in biological features such as the length composition and individual body condition of eastern Baltic G. morhua, should be accounted for when estimating stock reproductive potential.     

ArHsp40, a type 1 J-domain protein<Emphasis Type="Italic">,</Emphasis> is developmentally regulated and stress inducible in post-diapause <Emphasis Type="Italic">Artemia franciscana</Emphasis>

Upon diapause termination and exposure to favorable environmental conditions, cysts of the crustacean Artemia franciscana reinitiate development, a process dependent on the resumption of metabolic activity and the maintenance of protein homeostasis. The objective of the work described herein was to characterize molecular chaperones during post-diapause growth of A. franciscana. An Hsp40 complementary DNA (cDNA) termed ArHsp40 was cloned and shown to encode a protein with an amino-terminal J-domain containing a conserved histidine, proline, and aspartic acid (HPD) motif. Following the J-domain was a Gly/Phe (G/F) rich domain, a zinc-binding domain which contained a modified CXXCXGXG motif, and the carboxyl-terminal substrate binding region, all characteristics of type I Hsp40. Multiple alignment and protein modeling showed that ArHsp40 is comparable to Hsp40s from other eukaryotes and likely to be functionally similar. qRT-PCR revealed that during post-diapause development, ArHsp40 messenger RNA (mRNA) varied slightly until the E2/E3 stage and decreased significantly upon hatching. The immunoprobing of Western blots demonstrated that ArHsp40 was also relatively constant until E2/E3 and then declined dramatically. The drop in ArHsp40 when metabolism and protein synthesis were increasing was unexpected and demonstrated developmental regulation. The reduction in ArHsp40 at such an active life history stage indicates, as one possibility, that A. franciscana possesses additional Hsp40s, one or more of which replaces ArHsp40 as development progresses. Increased synthesis upon heat shock established that in addition to being developmentally regulated, ArHsp40 is stress inducible and, because it is found in mature cysts, ArHsp40 has the potential to contribute to stress tolerance during diapause.