SummaryCentrosome function in cell division requires their duplication, once, and only once, per cell cycle. Underlying centrosome duplication are alternating cycles of centriole assembly and separation [1]. Work in vertebrates has implicated the cysteine protease separase in anaphase-coupled centriole separation (or disengagement) and identified this as a key step in licensing another round of assembly [2]. Current models have separase cleaving a physical link between centrioles, potentially cohesin [3, 4], that prevents reinitiation of centriole assembly unless disengaged. Here, we examine separase function in the C. elegans early embryo. We find that depletion impairs separation and consequently duplication of sperm-derived centrioles at the meiosis-mitosis transition. However, subsequent cycles proceed normally. Whereas mitotic centrioles separate in the context of cortical forces acting on a disassembling pericentriolar material, sperm centrioles are not associated with significant pericentriolar material or subject to strong forces. Increasing centrosomal microtubule nucleation restores sperm centriole separation and duplication in separase-depleted embryos, while forced pericentriolar material disassembly drives premature separation in mitosis. These results emphasize the critical role of cytoskeletal forces and the pericentriolar material in centriole separation. Separase contributes to separation where forces are limited, offering a potential explanation for results obtained in different experimental models [5–7].

Among the 15 extracellular domains of the mannose 6-phosphate/ insulin-like growth factor-2 receptor (M6P/IGF2R), domain 11 has evolved a binding site for IGF2 to negatively regulate ligand bioavailability and mammalian growth. Despite the highly evolved structural loops of the IGF2:domain 11 binding site, affinity-enhancing AB loop mutations suggest that binding is modifiable. Here we examine the extent to which IGF2:domain 11 affinity, and its specificity over IGF1, can be enhanced, and we examine the structural basis of the mechanistic and functional consequences. Domain 11 binding loop mutants were selected by yeast surface display combined with high-resolution structure-based predictions, and validated by surface plasmon resonance. We discovered previously unidentified mutations in the ligand-interacting surface binding loops (AB, CD, FG, and HI). Five combined mutations increased rigidity of the AB loop, as confirmed by NMR. When added to three independently identified CD and FG loop mutations that reduced the k off value by twofold, these mutations resulted in an overall selective 100-fold improvement in affinity. The structural basis of the evolved affinity was improved shape complementarity established by interloop (AB-CD) and intraloop (FG-FG) side chain interactions. The high affinity of the combinatorial domain 11 Fc fusion proteins functioned as ligand-soluble antagonists or traps that depleted pathological IGF2 isoforms from serum and abrogated IGF2-dependent signaling in vivo. An evolved and reengineered high-specificity M6P/IGF2R domain 11 binding site for IGF2 may improve therapeutic targeting of the frequent IGF2 gain of function observed in human cancer.growth factor receptor | protein evolution | insulin-like growth factor 2 | binding kinetics | biological therapy T he functional evolution of proteins is largely considered to occur by chance, frequently because of unpredictable and specific events that confer a structure-based change in function sufficient for subsequent selection or "gain of fitness" (1). One such evolutionary biochemical example is the initial acquisition and subsequent gain of affinity between the insulin-like growth factor 2 (IGF2) ligand and a single domain of a nonsignaling mannose 6-phosphate (M6P)/IGF2 receptor (IGF2R) (domain 11). The structural and functional basis of this evolutionary path, which has occurred over 150 million years of mammalian evolution, has been reported previously (2). The questions that we address in the present work are whether the IGF2:domain 11 interaction has reached an optimal state in the context of IGF2 activation of signaling receptors and in the ligand clearance function of M6P/IGF2R, and how far can we extend the binding interaction in terms of structural, biophysical, and functional properties.Functionally, and unlike products of other mammalian imprinted genes, domain 11 is unusual because it specifically evolved to bind to an evolutionary conserved IGF2 ligand with high affinity (3-5). After binding, clearance of extracellular IGF2...

Patient-derived cancer 3D models are a promising tool that will revolutionize personalized cancer therapy but that require previous knowledge of optimal cell growth conditions and the most advantageous parameters to evaluate biomimetic relevance and monitor therapy efficacy. This study aims to establish general guidelines on 3D model characterization phenomena, focusing on neuroblastoma. We generated gelatin-based scaffolds with different stiffness and performed SK-N-BE(2) and SH-SY5Y aggressive neuroblastoma cell cultures, also performing co-cultures with mouse stromal Schwann cell line (SW10). Model characterization by digital image analysis at different time points revealed that cell proliferation, vitronectin production, and migration-related gene expression depend on growing conditions and are specific to the tumor cell line. Morphometric data show that 3D in vitro models can help generate optimal patient-derived cancer models, by creating, identifying, and choosing patterns of clinically relevant artificial microenvironments to predict patient tumor cell behavior and therapeutic responses.

The invasive tumor front (the tumor–host interface) is vitally important in malignant cell progression and metastasis. Tumor cell interactions with resident and infiltrating host cells and with the surrounding extracellular matrix and secreted factors ultimately determine the fate of the tumor. Herein we focus on the invasive tumor front, making an in-depth characterization of reticular fiber scaffolding, infiltrating immune cells, gene expression, and epigenetic profiles of classified aggressive primary uterine adenocarcinomas (24 patients) and leiomyosarcomas (11 patients). Sections of formalin-fixed samples before and after microdissection were scanned and studied. Reticular fiber architecture and immune cell infiltration were analyzed by automatized algorithms in colocalized regions of interest. Despite morphometric resemblance between reticular fibers and high presence of macrophages, we found some variance in other immune cell populations and distinctive gene expression and cell adhesion-related methylation signatures. Although no evident overall differences in immune response were detected at the gene expression and methylation level, impaired antimicrobial humoral response might be involved in uterine leiomyosarcoma spread. Similarities found at the invasive tumor front of uterine adenocarcinomas and leiomyosarcomas could facilitate the use of common biomarkers and therapies. Furthermore, molecular and architectural characterization of the invasive front of uterine malignancies may provide additional prognostic information beyond established prognostic factors.