Arp2/3 complex: advances on the inner workings of a molecular machine.

Several new papers report progress on the structure and function of Arp2/3 complex. A crystal structure, a cryo-EM structure, and a reconstitution of the complex from subunits have been reported. New results also address the nucleation mechanism and the role of bound nucleotide.     

Optimized inner ear organoids for efficient hair cell generation and ototoxicity response modeling

Hair cells in the mammalian cochlea are highly vulnerable to damage from drug toxicity, noise exposure, aging, and genetic mutations, with no capacity for regeneration. Progress in hair cell protection research has been limited by the scarcity of cochlear tissue and suitable in vitro models. Here, we present a novel one-step, self-organizing inner ear organoid system optimized with small molecules, which bypasses the need for multi-step expansion and forced differentiation protocols. This approach efficiently generates hair cells and supporting cells that recapitulate the molecular, cellular, and structural characteristics of the inner ear. Single-cell RNA sequencing revealed the diversity and fidelity of cell populations within the organoids. Utilizing this platform, we validated the protective effects of candidate compounds against hair cell damage, highlighting its potential as a powerful tool for drug discovery and mechanistic studies of hair cell protection.     

Exploring the human lacrimal gland using organoids and single-cell sequencing

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Tryptophan synthase: the workings of a channeling nanomachine

Substrate channeling between enzymes has an important role in cellular metabolism by compartmentalizing cytoplasmic synthetic processes. The bacterial tryptophan synthases are multienzyme nanomachines that catalyze the last two steps in L-tryptophan biosynthesis. The common metabolite indole is transferred from one enzyme to the other in each alphabeta-dimeric unit of the alpha2beta2 complex via an interconnecting 25-A-long tunnel. Recent solution studies of the Salmonella typhimurium alpha2beta2 complex coupled with X-ray crystal-structure determinations of complexes with substrates, intermediates and substrate analogs have driven important breakthroughs concerning the identification of the linkages between the bi-enzyme complex structure, catalysis at the alpha- and beta-active sites, and the allosteric regulation of substrate channeling.     

Flt-1-dependent survival characterizes the epithelial-mesenchymal transition of colonic organoids

Aberrant cell survival and resistance to apoptosis are hallmarks of tumor invasion and progression to metastatic disease, but the mechanisms involved are poorly understood. The epithelial-mesenchymal transition (EMT), a process that facilitates progression to invasive cancer, provides a superb model for studying such survival mechanisms. Here, we used a unique spheroid culture system that recapitulates the structure of the colonic epithelium and undergoes an EMT in response to cytokine stimulation to study this problem. Our data reveal that the EMT results in the increased expression of both VEGF and Flt-1, a tyrosine kinase VEGF receptor, and that the survival of these cells depends on a VEGF/Flt-1 autocrine pathway. Perturbation of Flt-1 function by either a blocking antibody or adenoviral expression of soluble Flt-1, which acts in a dominant-negative fashion, caused massive apoptosis only in cells that underwent EMT. This pathway was critical for the survival of other invasive colon carcinoma cell lines, and we observed a correlative upregulation of Flt-1 expression linked to in vivo human cancer progression. A role for Flt-1 in cell survival is unprecedented and has significant implications for Flt-1 function in tumor progression, as well as in other biological processes, including angiogenesis and development.     

Tumor necrosis factor-alpha stimulates the epithelial-to-mesenchymal transition of human colonic organoids

An epithelial-mesenchymal transition (EMT) characterizes the progression of many carcinomas and it is linked to the acquisition of an invasive phenotype. Given that the tumor microenvironment is an active participant in tumor progression, an important issue is whether a reactive stroma can modulate this process. Using a novel EMT model of colon carcinoma spheroids, we demonstrate that their transforming-growth factor-beta1 (TGF-beta)-induced EMT is accelerated dramatically by the presence of activated macrophages, and we identify tumor necrosis factor-alpha (TNF-alpha) as the critical factor produced by macrophages that accelerates the EMT. A synergy of TNF-alpha and TGF-beta signaling promotes a rapid morphological conversion of the highly organized colonic epithelium to dispersed cells with a mesenchymal phenotype, and this process is dependent on enhanced p38 MAPK activity. Moreover, exposure to TNF-alpha stimulates a rapid burst of ERK activation that results in the autocrine production of this cytokine by the tumor cells themselves. These results establish a novel role for the stroma in influencing EMT in colon carcinoma, and they identify a selective advantage to the stromal presence of infiltrating leukocytes in regulating malignant tumor progression.