Affinity fine-tuning anti-CAIX CAR-T cells mitigate on-target off-tumor side effects.

One of the major hurdles that has hindered the success of chimeric antigen receptor (CAR) T cell therapies against solid tumors is on-target off-tumor (OTOT) toxicity due to sharing of the same epitopes on normal tissues. To elevate the safety profile of CAR-T cells, an affinity/avidity fine-tuned CAR was designed enabling CAR-T cell activation only in the presence of a highly expressed tumor associated antigen (TAA) but not when recognizing the same antigen at a physiological level on healthy cells. Using direct stochastic optical reconstruction microscopy (dSTORM) which provides single-molecule resolution, and flow cytometry, we identified high carbonic anhydrase IX (CAIX) density on clear cell renal cell carcinoma (ccRCC) patient samples and low-density expression on healthy bile duct tissues.

Rapid Production of Nanoscale Liposomes Using a 3D-Printed Reactor-In-A-Centrifuge: Formulation, Characterisation, and Super-Resolution Imaging.

Nanoscale liposomes have been extensively researched and employed clinically for the delivery of biologically active compounds, including chemotherapy drugs and vaccines, offering improved pharmacokinetic behaviour and therapeutic outcomes. Traditional laboratory-scale production methods often suffer from limited control over liposome properties (e.g.

Molecular basis of MKLP2-dependent Aurora B transport from chromatin to the anaphase central spindle.

The Aurora B chromosomal passenger complex (CPC) is a conserved regulator of mitosis. Its functions require localization first to the chromosome arms and then centromeres in mitosis and subsequently the central spindle in anaphase. Here, we analyze the requirements for core CPC subunits, survivin and INCENP, and the mitotic kinesin-like protein 2 (MKLP2) in targeting to these distinct localizations.

Compound heterozygous loss-of-function mutations in KIF20A are associated with a novel lethal congenital cardiomyopathy in two siblings.

Congenital or neonatal cardiomyopathies are commonly associated with a poor prognosis and have multiple etiologies. In two siblings, a male and female, we identified an undescribed type of lethal congenital restrictive cardiomyopathy affecting the right ventricle. We hypothesized a novel autosomal recessive condition.

14-3-3 regulation of Ncd reveals a new mechanism for targeting proteins to the spindle in oocytes.

The meiotic spindle is formed without centrosomes in a large volume of oocytes. Local activation of crucial spindle proteins around chromosomes is important for formation and maintenance of a bipolar spindle in oocytes. We found that phosphodocking 14-3-3 proteins stabilize spindle bipolarity in oocytes.

A PP2A-B55 recognition signal controls substrate dephosphorylation kinetics during mitotic exit.

PP2A-B55 is one of the major phosphatases regulating cell division. Despite its importance for temporal control during mitotic exit, how B55 substrates are recognized and differentially dephosphorylated is unclear. Using phosphoproteomics combined with kinetic modeling to extract B55-dependent rate constants, we have systematically identified B55 substrates and assigned their temporal order in mitotic exit.

KIF4A and PP2A-B56 form a spatially restricted feedback loop opposing Aurora B at the anaphase central spindle.

The mitotic kinase Aurora B is concentrated at the anaphase central spindle by the kinesin MKlp2 during mitotic exit and cytokinesis. This pool of Aurora B phosphorylates substrates including the kinesin KIF4A to regulate central spindle length. In this paper, we identify a counteracting system in which PP2A-B56γ and -ε, but not PP2A-B56α, -β, and -δ, are maintained at the central spindle by KIF4A.

PP2A-B56 opposes Mps1 phosphorylation of Knl1 and thereby promotes spindle assembly checkpoint silencing.

The spindle assembly checkpoint (SAC) monitors correct attachment of chromosomes to microtubules, an important safeguard mechanism ensuring faithful chromosome segregation in eukaryotic cells. How the SAC signal is turned off once all the chromosomes have successfully attached to the spindle remains an unresolved question. Mps1 phosphorylation of Knl1 results in recruitment of the SAC proteins Bub1, Bub3, and BubR1 to the kinetochore and production of the wait-anaphase signal.

Rab18 and a Rab18 GEF complex are required for normal ER structure.

The ancestral Rab GTPase Rab18 and both subunits of the Rab3GAP complex are mutated in the human neurological and developmental disorder Warburg Micro syndrome. Here, we demonstrate that the Rab3GAP complex is a specific Rab18 guanine nucleotide exchange factor (GEF). The Rab3GAP complex localizes to the endoplasmic reticulum (ER) and is necessary for ER targeting of Rab18.

Diversity and plasticity in Rab GTPase nucleotide release mechanism has consequences for Rab activation and inactivation.

Ras superfamily GTPase activation and inactivation occur by canonical nucleotide exchange and GTP hydrolysis mechanisms. Despite conservation of active-site residues, the Ras-related Rab GTPase activation pathway differs from Ras and between different Rabs. Analysis of DENND1-Rab35, Rabex-Rab5, TRAPP-Rab1 and DrrA-Rab1 suggests Rabs have the potential for activation by distinct GDP-release pathways.

The BEG (PP2A-B55/ENSA/Greatwall) pathway ensures cytokinesis follows chromosome separation.

Cytokinesis follows separase activation and chromosome segregation. This order is ensured in budding yeast by the mitotic exit network (MEN), where Cdc14p dephosphorylates key conserved Cdk1-substrates exemplified by the anaphase spindle-elongation protein Ase1p. However, in metazoans, MEN and Cdc14 function is not conserved.

Aurora B suppresses microtubule dynamics and limits central spindle size by locally activating KIF4A.

Anaphase central spindle formation is controlled by the microtubule-stabilizing factor PRC1 and the kinesin KIF4A. We show that an MKlp2-dependent pool of Aurora B at the central spindle, rather than global Aurora B activity, regulates KIF4A accumulation at the central spindle. KIF4A phosphorylation by Aurora B stimulates the maximal microtubule-dependent ATPase activity of KIF4A and promotes its interaction with PRC1.

Melanoma-associated mutations in protein phosphatase 6 cause chromosome instability and DNA damage owing to dysregulated Aurora-A.

Mutations in the PPP6C catalytic subunit of protein phosphatase 6 (PP6) are drivers for the development of melanoma. Here, we analyse a panel of melanoma-associated mutations in PPP6C and find that these generally compromise assembly of the PP6 holoenzyme and catalytic activity towards a model substrate. Detailed analysis of one mutant, PPP6C-H114Y, in both primary melanoma and engineered cell lines reveals it is destabilized and undergoes increased proteasome-mediated turnover.

CYK4 inhibits Rac1-dependent PAK1 and ARHGEF7 effector pathways during cytokinesis.

In mitosis, animal cells lose their adhesion to the surrounding surfaces and become rounded. During mitotic exit, they reestablish these adhesions and at the same time physically contract and divide. How these competing processes are spatially segregated at the cell cortex remains mysterious.

Rab14 and its exchange factor FAM116 link endocytic recycling and adherens junction stability in migrating cells.

Rab GTPases define the vesicle trafficking pathways underpinning cell polarization and migration. Here, we find that Rab4, Rab11, and Rab14 and the candidate Rab GDP-GTP exchange factors (GEFs) FAM116A and AVL9 are required for cell migration. Rab14 and its GEF FAM116A localize to and act on an intermediate compartment of the transferrin-recycling pathway prior to Rab11 and after Rab5 and Rab4.

Loss-of-function mutations in RAB18 cause Warburg micro syndrome.

Warburg Micro syndrome and Martsolf syndrome are heterogenous autosomal-recessive developmental disorders characterized by brain, eye, and endocrine abnormalities. Previously, identification of mutations in RAB3GAP1 and RAB3GAP2 in both these syndromes implicated dysregulation of the RAB3 cycle (which controls calcium-mediated exocytosis of neurotransmitters and hormones) in disease pathogenesis. RAB3GAP1 and RAB3GAP2 encode the catalytic and noncatalytic subunits of the hetrodimeric enzyme RAB3GAP (RAB3GTPase-activating protein), a key regulator of the RAB3 cycle.

Protein phosphatase 6 regulates mitotic spindle formation by controlling the T-loop phosphorylation state of Aurora A bound to its activator TPX2.

Many protein kinases are activated by a conserved regulatory step involving T-loop phosphorylation. Although there is considerable focus on kinase activator proteins, the importance of specific T-loop phosphatases reversing kinase activation has been underappreciated. We find that the protein phosphatase 6 (PP6) holoenzyme is the major T-loop phosphatase for Aurora A, an essential mitotic kinase.

Plk1 negatively regulates Cep55 recruitment to the midbody to ensure orderly abscission.

Cytokinesis requires a membrane-remodeling and fission event termed abscission that occurs after chromosome segregation, cleavage furrow formation, and contraction have completed. In this study, we show how abscission factor recruitment is controlled by the Polo-like kinase 1 (Plk1). At the metaphase-anaphase transition, Plk1 initiates cleavage furrow formation and is then progressively degraded during mitotic exit.

Production of heterologous proteins in yeast with the aid of the Hsp150delta carrier.

Proper folding, and consequently exit from the endoplasmic reticulum (ER) and secretion of heterologous exocytic proteins in yeast can be rescued by fusing the proteins to certain yeast-derived polypeptides. Biologically active mammalian glycoproteins can be produced in Saccharomyces cerevisiae and Pichia pastoris by joining them to a fragment of a natural secretory glycoprotein of S. cerevisiae, Hsp150delta.

Endoplasmic reticulum exit of a secretory glycoprotein in the absence of sec24p family proteins in yeast.

Glycoproteins exit the endoplasmic reticulum (ER) of the yeast Saccharomyces cerevisiae in coat protein complex II (COPII) coated vesicles. The coat consists of the essential proteins Sec23p, Sec24p, Sec13p, Sec31p, Sar1p and Sec16p. Sec24p and its two nonessential homologues Sfb2p and Sfb3p have been suggested to serve in cargo selection.