Probing the dynamic crosstalk of lysosomes and mitochondria with structured illumination microscopy.

Structured illumination microscopy (SIM) is a super-resolution technology for imaging living cells and has been used for studying the dynamics of lysosomes and mitochondria. Recently, new probes and analyzing methods have been developed for SIM imaging, enabling the quantitative analysis of these subcellular structures and their interactions. This review provides an overview of the working principle and advances of SIM, as well as the organelle-targeting principles and types of fluorescence probes, including small molecules, metal complexes, nanoparticles, and fluorescent proteins. Additionally, quantitative methods based on organelle morphology and distribution are outlined. Finally, the review provides an outlook on the current challenges and future directions for improving the combination of SIM imaging and image analysis to further advance the study of organelles. We hope that this review will be useful for researchers working in the field of organelle research and help to facilitate the development of SIM imaging and analysis techniques.

Mitochondrial nucleoid condensates drive peripheral fission through high membrane curvature.

Mitochondria are dynamic organelles that undergo fusion and fission events, in which the mitochondrial membrane and DNA (mtDNA) play critical roles. The spatiotemporal organization of mtDNA reflects and impacts mitochondrial dynamics. Herein, to study the detailed dynamics of mitochondrial membrane and mtDNA, we rationally develop a dual-color fluorescent probe, mtGLP, that could be used for simultaneously monitoring mitochondrial membrane and mtDNA dynamics via separate color outputs. By combining mtGLP with structured illumination microscopy to monitor mitochondrial dynamics, we discover the formation of nucleoid condensates in damaged mitochondria. We further reveal that nucleoid condensates promoted the peripheral fission of damaged mitochondria via asymmetric segregation. Through simulations, we find that the peripheral fission events occurred when the nucleoid condensates interacted with the highly curved membrane regions at the two ends of the mitochondria. Overall, we show that mitochondrial nucleoid condensates utilize peripheral fission to maintain mitochondrial homeostasis.

Advanced imaging techniques for tracking drug dynamics at the subcellular level.

Optical microscopes are an important imaging tool that have effectively advanced the development of modern biomedicine. In recent years, super-resolution microscopy (SRM) has become one of the most popular techniques in the life sciences, especially in the field of living cell imaging. SRM has been used to solve many problems in basic biological research and has great potential in clinical application. In particular, the use of SRM to study drug delivery and kinetics at the subcellular level enables researchers to better study drugs' mechanisms of action and to assess the efficacy of their targets in vivo. The purpose of this paper is to review the recent advances in SRM and to highlight some of its applications in assessing subcellular drug dynamics.

Characterization of the Key Determinants of Phd Antitoxin Mediated Doc Toxin Inactivation in Salmonella.

In the search for novel antimicrobial therapeutics, toxin-antitoxin (TA) modules are promising yet underexplored targets for overcoming antibiotic failure. The bacterial toxin Doc has been associated with the persistence of Salmonella in macrophages, enabling its survival upon antibiotic exposure. After developing a novel method to produce the recombinant toxin, we have used antitoxin-mimicking peptides to thoroughly investigate the mechanism by which its cognate antitoxin Phd neutralizes the activity of Doc. We reveal insights into the molecular detail of the Phd-Doc relationship and discriminate antitoxin residues that stabilize the TA complex from those essential for inhibiting the activity of the toxin. Coexpression of Doc and antitoxin peptides in Salmonella was able to counteract the activity of the toxin, confirming our in vitro results with equivalent sequences. Our findings provide key principles for the development of chemical tools to study and therapeutically interrogate this important class of protein-protein interactions.

Diverse functionalization of Aurora-A kinase at specified surface and buried sites by native chemical modification.

The ability to obtain a homogeneous sample of protein is invaluable when studying the effect of alterations such as post-translational modifications (PTMs). Selective functionalization of a protein to investigate the effect of PTMs on its structure or activity can be achieved by chemical modification of cysteine residues. We demonstrate here that one such technique, which involves conversion of cysteine to dehydroalanine followed by thiol nucleophile addition, is suitable for the site-specific installation of a wide range of chemical mimics of PTMs, including acetylated and dimethylated lysine, and other unnatural amino acids. These reactions, optimized for the clinically relevant kinase Aurora-A, readily proceed to completion as revealed by intact protein mass spectrometry. Moreover, these reactions proceed under non-denaturing conditions, which is desirable when working with large protein substrates. We have determined reactivity trends for a diverse range of thiol nucleophile addition reactions at two separate sites on Aurora-A, and we also highlight limitations when using thiol nucleophiles that contain basic functional groups. We show that chemical modification of cysteine residues is possible not only on a flexible surface-exposed loop, but also within a deep active site pocket at the conserved DFG motif, which reveals the potential use of this method in exploring enzyme function through modification of catalytic site residues.

Insights into Aurora-A kinase activation using unnatural amino acids incorporated by chemical modification.

Most protein kinases are regulated through activation loop phosphorylation, but the contributions of individual sites are largely unresolved due to insufficient control over sample phosphorylation. Aurora-A is a mitotic Ser/Thr protein kinase that has two regulatory phosphorylation sites on its activation loop, T287 and T288. While phosphorylation of T288 is known to activate the kinase, the function of T287 phosphorylation is unclear. We applied site-directed mutagenesis and selective chemical modification to specifically introduce bioisosteres for phospho-threonine and other unnatural amino acids at these positions. Modified Aurora-A proteins were characterized using a biochemical assay measuring substrate phosphorylation. Replacement of T288 with glutamate and aspartate weakly stimulated activity. Phospho-cysteine, installed by chemical synthesis from a corresponding cysteine residue introduced at position 288, showed catalytic activity approaching that of the comparable phospho-serine protein. Unnatural amino acid residues, with longer side chains, inserted at position 288 were autophosphorylated and supported substrate phosphorylation. Aurora-A activity is enhanced by phosphorylation at position 287 alone but is suppressed when position 288 is also phosphorylated. This is rationalized by competition between phosphorylated T287 and T288 for a binding site composed of arginines, based on a structure of Aurora-A in which phospho-T287 occupies this site. This is, to our knowledge, the first example of a Ser/Thr kinase whose activity is controlled by the phosphorylation state of adjacent residues in its activation loop. Overall we demonstrate an approach that combines mutagenesis and selective chemical modification of selected cysteine residues to investigate otherwise impenetrable aspects of kinase regulation.

Design of potent and selective hybrid inhibitors of the mitotic kinase Nek2: structure-activity relationship, structural biology, and cellular activity.

We report herein a series of Nek2 inhibitors based on an aminopyridine scaffold. These compounds have been designed by combining key elements of two previously discovered chemical series. Structure based design led to aminopyridine (R)-21, a potent and selective inhibitor able to modulate Nek2 activity in cells.

Antibody removal before ABO-incompatible renal transplantation: how much plasma exchange is therapeutic?

BACKGROUND: ABOi transplantation is an accepted method of expanding the kidney donor pool but there is little analysis of the protocols used. We established an ABOi programme utilising leukocyte depletion, tacrolimus, TPE and IvIg. There are few reports in the literature on the success rates of antibody removal protocols or relating to patients in whom antibody removal fails. The purpose of this study was to define the likelihood of achieving transplantation depending on ABO antibody titers. METHODS: 56 patients entered our ABOi program. Data were analysed to determine the likelihood of achieving transplantation, ABO antibody titre prior to antibody removal and amount of TPE required to achieve transplantation. The median antibody titer was 1:64 (Range 0-1:1024). Transplantation proceeded when the ABO titer reached ≤1:4. RESULTS: 51/56 (91%) patients achieved transplantation after 8.3±5 TPE. Five patients with high ABO titers were not transplanted despite extensive TPE. The number of TPE required to reach an ABO titer of ≤1:4 correlates best with pre-treatment IgG titers. CONCLUSIONS: This is the first study to demonstrate a cut off titer for entry in to the ABO incompatible program using the relationship between ABO titer and amount of TPE required to reach transplantation. We now tailor the antibody removal protocol prior to transplantation and have introduced a cut-off entry titer to the program (≤1:256), because of the unacceptable risk of exposing patients with higher titers to long-lasting immunosuppression and costly, prolonged, courses of TPE without the guarantee of successful transplantation. Patients whose ABO titer exceeds the cut-off are counselled and offered alternative routes to transplantation.

Expression, crystallization and preliminary X-ray data analysis of NT-Als9-2, a fungal adhesin from Candida albicans.

Candida albicans is a common human fungal commensal that can also cause a range of infections from skin/mucosal `thrush' to severe systemic candidiasis. Adherence to host cells is one of the key determinants of Candida pathogenesis. The Als family of surface proteins has been implicated in adhesion of C. albicans, yet limited information has been published on the structure and mechanism of these fungal adhesins. The N-terminal region of these proteins has been shown to possess adhesive properties, making it a possible target for new therapeutic strategies. Recombinant NT-Als9-2 from C. albicans (residues 18-329) was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.0 Šresolution. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 34.73, b = 68.71, c = 120.03 Å, α = ß = γ = 90° and one molecule in the asymmetric unit. Platinum-derivatized crystals belonged to the same space group, with similar unit-cell parameters, although they were not completely isomorphous.