The use of quantitative imaging to investigate regulators of membrane trafficking in Arabidopsis stomatal closure

Expansion of gene families facilitates robustness and evolvability of biological processes but impedes functional genetic dissection of signalling pathways. To address this, quantitative analysis of single cell responses can help characterize the redundancy within gene families. We developed high‐throughput quantitative imaging of stomatal closure, a response of plant guard cells, and performed a reverse genetic screen in a group of Arabidopsis mutants to five stimuli. Focussing on the intersection between guard cell signalling and the endomembrane system, we identified eight clusters based on the mutant stomatal responses. Mutants generally affected in stomatal closure were mostly in genes encoding SNARE and SCAMP membrane regulators. By contrast, mutants in RAB5 GTPase genes played specific roles in stomatal closure to microbial but not drought stress. Together with timed quantitative imaging of endosomes revealing sequential patterns in FLS2 trafficking, our imaging pipeline can resolve non‐redundant functions of the RAB5 GTPase gene family. Finally, we provide a valuable image‐based tool to dissect guard cell responses and outline a genetic framework of stomatal closure.      

Cryopreservation of composite tissues and transplantation: preliminary studies

Despite advances in cryobiology, the reliable cryopreservation of complex tissues has not yet been achieved. This study evaluates the viability of cryopreserved composite flaps and demonstrates the feasibility of their transplantation. Epigastric flaps were harvested from male Lewis rats. 1.5 M dimethyl sulfoxide (Me₂SO) was used as the initial cryoprotectant agent (CPA). Samples were frozen at controlled rate to −140 °C and transferred to liquid nitrogen for at least two weeks. Hematoxylin and eosin (H/E) staining, MTT tetrazolium salt assay, and factor VIII immunostaining were used to evaluate the overall histology, epithelial viability, and vascular endothelial integrity, respectively, of cryopreserved flaps. For the in vivo phase, flaps were isotransplanted to 35 recipient animals, divided into three groups: fresh (n = 10), perfused (n = 8), and cryopreserved (n = 17). Blood vessel patency was assessed via Doppler at 1, 7, and 60 days post-transplantation. For in vitro studies, cryopreserved samples (10/10) retained normal cell architecture and vascular endothelial integrity upon H/E and factor VIII staining. The viability index of cryopreserved composite flap skin (n = 10) was 11.17 ± 2.01, which was not significantly different from fresh controls (n = 10, 12.15 ± 1.32). All transplanted flaps in the fresh and perfusion groups survived with healthy color and hair growth at 60 days after operation. Survival in the cryopreserved group ranged from 2 to 60 days, with a mean of 12 days. These results demonstrate that the long term survival of cryopreserved composite tissue transplants is possible. Further studies are needed to refine protocols for the reliable cryopreservation of composite parts.     

Generation and characterization of a functional human adipose-derived multipotent mesenchymal stromal cell line

Multipotent mesenchymal stromal cells (MSC) and MSC-derived products have emerged as promising therapeutic tools. To fully exploit their potential, further mechanistic studies are still necessary and bioprocessing needs to be optimized, which requires an abundant supply of functional MSC for basic research. To address this need, here we used a novel technology to establish a human adipose-derived MSC line with functional characteristics representative of primary MSC. Primary MSC were isolated and subjected to lentiviral transduction with a library of expansion genes. Clonal cell lines were generated and evaluated on the basis of their morphology, immunophenotype, and proliferation potential. One clone (K5 iMSC) was then selected for further characterization. This clone had integrated a specific transgene combination including genes involved in stemness and maintenance of adult stem cells. Favorably, the K5 iMSC showed cell characteristics resembling juvenile MSC, as they displayed a shorter cell length and enhanced migration and proliferation compared with the non-immortalized original primary MSC (p < 0.05). Still, their immunophenotype and differentiation potential corresponded to the original primary MSC and the MSC definition criteria, and cytogenetic analyses revealed no clonal aberrations. We conclude that the technology used is applicable to generate functional MSC lines for basic research and possible future bioprocessing applications.     

Partially folded intermediates as critical precursors of light chain amyloid fibrils and amorphous aggregates

Light chain, or AL, amyloidosis is a pathological condition arising from systemic extracellular deposition of monoclonal immunoglobulin light chain variable domains in the form of insoluble amyloid fibrils, especially in the kidneys. Substantial evidence suggests that amyloid fibril formation from native proteins occurs via a conformational change leading to a partially folded intermediate conformation, whose subsequent association is a key step in fibrillation. In the present investigation, we have examined the properties of a recombinant amyloidogenic light chain variable domain, SMA, to determine whether partially folded intermediates can be detected and correlated with aggregation. The results from spectroscopic and hydrodynamic measurements, including far- and near-UV circular dichroism, FTIR, NMR, and intrinsic tryptophan fluorescence and small-angle X-ray scattering, reveal the build-up of two partially folded intermediate conformational states as the pH is decreased (low pH destabilized the protein and accelerated the kinetics of aggregation). A relatively nativelike intermediate, I(N), was observed between pH 4 and 6, with little loss of secondary structure, but with significant tertiary structure changes and enhanced ANS binding, indicating exposed hydrophobic surfaces. At pH below 3, we observed a relatively unfolded, but compact, intermediate, I(U), which was characterized by decreased tertiary and secondary structure. The I(U) intermediate readily forms amyloid fibrils, whereas I(N) preferentially leads to amorphous aggregates. Except at pH 2, where negligible amorphous aggregate is formed, the amorphous aggregates formed significantly more rapidly than the fibrils. This is the first indication that different partially folded intermediates may be responsible for different aggregation pathways (amorphous and fibrillar). The data support the hypothesis that amyloid fibril formation involves the ordered self-assembly of partially folded species that are critical soluble precursors of fibrils.     

Conservative duplication of spindle poles during meiosis in Saccharomyces cerevisiae

During sporulation in diploid Saccharomyces cerevisiae, spindle pole bodies acquire the so-called meiotic plaque, a prerequisite for spore formation. Mpc70p is a component of the meiotic plaque and is thus essential for spore formation. We show here that MPC70/mpc70 heterozygous strains most often produce two spores instead of four and that these spores are always nonsisters. In wild-type strains, Mpc70p localizes to all four spindle pole bodies, whereas in MPC70/mpc70 strains Mpc70p localizes to only two of the four spindle pole bodies, and these are always nonsisters. Our data can be explained by conservative spindle pole body distribution in which the two newly synthesized meiosis II spindle pole bodies of MPC70/mpc70 strains lack Mpc70p.     

Effect of environmental factors on the kinetics of insulin fibril formation: elucidation of the molecular mechanism

In the search for the molecular mechanism of insulin fibrillation, the kinetics of insulin fibril formation were studied under different conditions using the fluorescent dye thioflavin T (ThT). The effect of insulin concentration, agitation, pH, ionic strength, anions, seeding, and addition of 1-anilinonaphthalene-8-sulfonic acid (ANS), urea, TMAO, sucrose, and ThT on the kinetics of fibrillation was investigated. The kinetics of the fibrillation process could be described by the lag time for formation of stable nuclei (nucleation) and the apparent rate constant for the growth of fibrils (elongation). The addition of seeds eliminated the lag phase. An increase in insulin concentration resulted in shorter lag times and faster growth of fibrils. Shorter lag times and faster growth of fibrils were seen at acidic pH versus neutral pH, whereas an increase in ionic strength resulted in shorter lag times and slower growth of fibrils. There was no clear correlation between the rate of fibril elongation and ionic strength. Agitation during fibril formation attenuated the effects of insulin concentration and ionic strength on both lag times and fibril growth. The addition of ANS increased the lag time and decreased the apparent growth rate for insulin fibril formation. The ANS-induced inhibition appears to reflect the formation of amorphous aggregates. The denaturant, urea, decreased the lag time, whereas the stabilizers, trimethylamine N-oxide dihydrate (TMAO) and sucrose, increased the lag times. The results indicated that both nucleation and fibril growth were controlled by hydrophobic and electrostatic interactions. A kinetic model, involving the association of monomeric partially folded intermediates, whose concentration is stimulated by the air-water interface, leading to formation of the critical nucleus and thence fibrils, is proposed.     

Morphological control of inositol-1,4,5-trisphosphate-dependent signals

Inositol-1,4,5-trisphosphate (InsP(3))-mediated calcium signals represent an important mechanism for transmitting external stimuli to the cell. However, information about intracellular spatial patterns of InsP(3) itself is not generally available. In particular, it has not been determined how the interplay of InsP(3) generation, diffusion, and degradation within complex cellular geometries can control the patterns of InsP(3) signaling. Here, we explore the spatial and temporal characteristics of [InsP(3)](cyt) during a bradykinin-induced calcium wave in a neuroblastoma cell. This is achieved by using a unique image-based computer modeling system, Virtual Cell, to integrate experimental data on the rates and spatial distributions of the key molecular components of the process. We conclude that the characteristic calcium dynamics requires rapid, high-amplitude production of [InsP(3)](cyt) in the neurite. This requisite InsP(3) spatiotemporal profile is provided, in turn, as an intrinsic consequence of the cell's morphology, demonstrating how geometry can locally and dramatically intensify cytosolic signals that originate at the plasma membrane. In addition, the model predicts, and experiments confirm, that stimulation of just the neurite, but not the soma or growth cone, is sufficient to generate a calcium response throughout the cell.     

Unconventional myosins at the crossroad of signal transduction and cytoskeleton remodeling

The cytoplasm of eukaryotic cells is a complex milieu and unraveling how its unique cytoarchitecture is achieved and maintained is a central theme in modern cell biology. The actin cytoskeleton is essential for the maintenance of cell shape and locomotion, and also provides tracks for active intracellular transport. Myosins, the actin-dependent motor proteins form a superfamily of at least 15 structural classes and have been identified in a wide variety of organisms, making the presence of actin and myosins a hallmark feature of eukaryotes. Direct connections of myosins to a variety of cellular tasks are now emerging, such as in cytokinesis, phagocytosis, endocytosis, polarized secretion and exocytosis, axonal transport. Recent studies reveal that myosins also play an essential role in many aspects of signal transduction and neurosensation.     

Natively unfolded human prothymosin alpha adopts partially folded collapsed conformation at acidic pH.

Prothymosin alpha has previously been shown to be unfolded at neutral pH, thus belonging to a growing family of "natively unfolded" proteins. The structural properties and conformational stability of recombinant human prothymosin alpha were characterized at neutral and acidic pH by gel filtration, SAXS, circular dichroism, ANS fluorescence, (1)H NMR, and resistance to urea-induced unfolding. Interestingly, prothymosin alpha underwent a cooperative transition from the unfolded state into a partially folded conformation on lowering the pH. This conformation of prothymosin alpha is a compact denatured state, with structural properties different from those of the molten globule. The formation of alpha-helical structure by the glutamic acid-rich elements of the protein accompanied by the partial hydrophobic collapse is expected at lower pH due to the neutralization of the negatively charged residues. It is possible that such conformational changes may be associated with the protein function.