Centrosomal organization of Cep152 provides flexibility in Plk4 and procentriole positioning.

Centriole duplication is a high-fidelity process driven by Polo-like kinase 4 (Plk4) and a few conserved initiators. Dissecting how Plk4 and its receptors organize within centrosomes is critical to understand the centriole duplication process and biochemical and architectural differences between centrosomes of different species. Here, at nanoscale resolution, we dissect centrosomal localization of Plk4 in G1 and S phase in its catalytically active and inhibited state during centriole duplication and amplification. We build a precise distribution map of Plk4 and its receptor Cep152, as well as Cep44, Cep192, and Cep152-anchoring factors Cep57 and Cep63. We find that Cep57, Cep63, Cep44, and Cep192 localize in ninefold symmetry. However, during centriole maturation, Cep152, which we suggest is the major Plk4 receptor, develops a more complex pattern. We propose that the molecular arrangement of Cep152 creates flexibility for Plk4 and procentriole placement during centriole initiation. As a result, procentrioles form at variable positions in relation to the mother centriole microtubule triplets.

CPAP insufficiency leads to incomplete centrioles that duplicate but fragment.

Centrioles are structures that assemble centrosomes. CPAP is critical for centrosome assembly, and its mutations are found in patients with diseases such as primary microcephaly. CPAP's centrosomal localization, its dynamics, and the consequences of its insufficiency in human cells are poorly understood. Here we use human cells genetically engineered for fast degradation of CPAP, in combination with superresolution microscopy, to address these uncertainties. We show that three independent centrosomal CPAP populations are dynamically regulated during the cell cycle. We confirm that CPAP is critical for assembly of human centrioles, but not for recruitment of pericentriolar material on already assembled centrioles. Further, we reveal that CPAP insufficiency leads to centrioles with incomplete microtubule triplets that can convert to centrosomes, duplicate, and form mitotic spindle poles, but fragment owing to loss of cohesion between microtubule blades. These findings further our basic understanding of the role of CPAP in centrosome biogenesis and help understand how CPAP aberrations can lead to human diseases.

Dynamin regulates the dynamics and mechanical strength of the actin cytoskeleton as a multifilament actin-bundling protein.

The dynamin GTPase is known to bundle actin filaments, but the underlying molecular mechanism and physiological relevance remain unclear. Our genetic analyses revealed a function of dynamin in propelling invasive membrane protrusions during myoblast fusion in vivo. Using biochemistry, total internal reflection fluorescence microscopy, electron microscopy and cryo-electron tomography, we show that dynamin bundles actin while forming a helical structure. At its full capacity, each dynamin helix captures 12-16 actin filaments on the outer rim of the helix. GTP hydrolysis by dynamin triggers disassembly of fully assembled dynamin helices, releasing free dynamin dimers/tetramers and facilitating Arp2/3-mediated branched actin polymerization. The assembly/disassembly cycles of dynamin promote continuous actin bundling to generate mechanically stiff actin super-bundles. Super-resolution and immunogold platinum replica electron microscopy revealed dynamin along actin bundles at the fusogenic synapse. These findings implicate dynamin as a unique multifilament actin-bundling protein that regulates the dynamics and mechanical strength of the actin cytoskeletal network.

Prolonged mitosis results in structurally aberrant and over-elongated centrioles.

Centrioles are precisely built microtubule-based structures that assemble centrosomes and cilia. Aberrations in centriole structure are common in tumors, yet how these aberrations arise is unknown. Analysis of centriole structure is difficult because it requires demanding electron microscopy. Here we employ expansion microscopy to study the origins of centriole structural aberrations in large populations of human cells. We discover that centrioles do not have an elongation monitoring mechanism, which renders them prone to over-elongation, especially during prolonged mitosis induced by various factors, importantly including supernumerary centrioles. We identify that mitotic centriole over-elongation is dependent on mitotic Polo-like kinase 1, which we uncover as a novel regulator of centriole elongation in human cycling cells. While insufficient Plk1 levels lead to the formation of shorter centrioles lacking a full set of microtubule triplets, its overactivity results in over-elongated and structurally aberrant centrioles. Our data help explain the origin of structurally aberrant centrioles and why centriole numerical and structural defects coexist in tumors.

Synaptojanin and Endophilin Mediate Neck Formation during Ultrafast Endocytosis.

Ultrafast endocytosis generates vesicles from the plasma membrane as quickly as 50 ms in hippocampal neurons following synaptic vesicle fusion. The molecular mechanism underlying the rapid maturation of these endocytic pits is not known. Here we demonstrate that synaptojanin-1, and its partner endophilin-A, function in ultrafast endocytosis. In the absence of synaptojanin or endophilin, the membrane is rapidly invaginated, but pits do not become constricted at the base. The 5-phosphatase activity of synaptojanin is involved in formation of the neck, but 4-phosphatase is not required. Nevertheless, these pits are eventually cleaved into vesicles; within a 30-s interval, synaptic endosomes form and are resolved by clathrin-mediated budding. Then synaptojanin and endophilin function at a second step to aid with the removal of clathrin coats from the regenerated vesicles. These data together suggest that synaptojanin and endophilin can mediate membrane remodeling on a millisecond timescale during ultrafast endocytosis.

MicroRNA-21 inhibits Serpini1, a gene with novel tumour suppressive effects in gastric cancer.

BACKGROUND: A thorough understanding of gastric cancer at the molecular level is urgently needed. One prominent oncogenic microRNA, miR-21, was previously reported to be upregulated in gastric cancer. METHODS: We performed an unbiased search for downstream messenger RNA targets of miR-21, based on miR-21 dysregulation, by using human tissue specimens and the MKN28 human gastric carcinoma cell line. Molecular techniques include microRNA microarrays, cDNA microarrays, qRT-PCR for miR and mRNA expression, transfection of MKN28 with miR-21 inhibitor or Serpini1 followed by Western blotting, cell cycle analysis by flow cytometry and luciferase reporter assay. RESULTS: This search identified Serpini1 as a putative miR-21 target. Luciferase assays demonstrated direct interaction between miR-21 and Serpini1 3'UTR. miR-21 and Serpini1 expression levels were inversely correlated in a subgroup of gastric cancers, suggesting a regulatory mechanism that included both of these molecules. Furthermore, Serpini1 induced growth retardation of MKN28 and induced vigorous G1/S arrest suggesting its potential tumour-suppressive function in the stomach. CONCLUSION: Taken together, these data suggest that in a subgroup of gastric cancers, miR-21 is upregulated, inducing downregulation of Serpini1, which in turn releases the G1-S transition checkpoint, with the end result being increased tumour growth.

MicroRNA down-regulated in human cholangiocarcinoma control cell cycle through multiple targets involved in the G1/S checkpoint.

UNLABELLED: MicroRNAs (miRs) recently emerged as prominent regulators of cancer processes. In the current study we aimed at elucidating regulatory pathways and mechanisms through which miR-494, one of the miR species found to be down-regulated in cholangiocarcinoma (CCA), participates in cancer homeostasis. miR-494 was identified as down-regulated in CCA based on miR arrays. Its expression was verified with quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). To enforce miR expression, we employed both transfection methods, as well as a retroviral construct to stably overexpress miR-494. Up-regulation of miR-494 in cancer cells decreased growth, consistent with a functional role. mRNA arrays of cells treated with miR-494, followed by pathway analysis, suggested that miR-494 impacts cell cycle regulation. Cell cycle analyses demonstrated that miR-494 induces a significant G1/S checkpoint reinforcement. Further analyses demonstrated that miR-494 down-regulates multiple molecules involved in this transition checkpoint. Luciferase reporter assays demonstrated a direct interaction between miR-494 and the 3'-untranslated region of cyclin-dependent kinase 6 (CDK6). Last, xenograft experiments demonstrated that miR-494 induces a significant cancer growth retardation in vivo. CONCLUSION: Our findings demonstrate that miR-494 is down-regulated in CCA and that its up-regulation induces cancer cell growth retardation through multiple targets involved in the G1-S transition. These findings support the paradigm that miRs are salient cellular signaling pathway modulators, and thus represent attractive therapeutic targets. miR-494 emerges as an important regulator of CCA growth and its further study may lead to the development of novel therapeutics.

Novel candidate colorectal cancer biomarkers identified by methylation microarray-based scanning.

DNA hypermethylation is a common epigenetic abnormality in colorectal cancers (CRCs) and a promising class of CRC screening biomarkers. We conducted a genome-wide search for novel neoplasia-specific hypermethylation events in the colon. We applied methylation microarray analysis to identify loci hypermethylated in 17 primary CRCs relative to eight non-neoplastic colonic mucosae (NCs) from neoplasia-free subjects. These CRC-associated hypermethylation events were then individually evaluated for their ability to discriminate neoplastic from non-neoplastic cases, based on real-time quantitative methylation-specific PCR (qMSP) assays in 113 colonic tissues: 51 CRCs, nine adenomas, 19 NCs from CRC patients (CRC-NCs), and 34 NCs from neoplasia-free subjects (control NCs). A strict microarray data filtering identified 169 candidate CRC-associated hypermethylation events. Fourteen of these 169 loci were evaluated using qMSP assays. Ten of these 14 methylation events significantly distinguished CRCs from age-matched control NCs (P<0.05 by receiver operator characteristic curve analysis); methylation of visual system homeobox 2 (VSX2) achieved the highest discriminative accuracy (83.3% sensitivity and 92.3% specificity, P<1×10(-6)), followed by BEN domain containing 4 (BEND4), neuronal pentraxin I (NPTX1), ALX homeobox 3 (ALX3), miR-34b, glucagon-like peptide 1 receptor (GLP1R), BTG4, homer homolog 2 (HOMER2), zinc finger protein 583 (ZNF583), and gap junction protein, gamma 1 (GJC1). Adenomas were significantly discriminated from control NCs by hypermethylation of VSX2, BEND4, NPTX1, miR-34b, GLP1R, and HOMER2 (P<0.05). CRC-NCs were significantly distinguished from control NCs by methylation of ALX3 (P<1×10(-4)). In conclusion, systematic methylome-wide analysis has identified ten novel methylation events in neoplastic and non-neoplastic colonic mucosae from CRC patients. These potential biomarkers significantly discriminate CRC patients from controls. Thus, they merit further evaluation in stool- and circulating DNA-based CRC detection studies.

Enzymatically triggered actuation of miniaturized tools.

We demonstrate a methodology that utilizes the specificity of enzyme-substrate biomolecular interactions to trigger miniaturized tools under biocompatible conditions. Miniaturized grippers were constructed using multilayer hinges that employed intrinsic strain energy and biopolymer triggers, as well as ferromagnetic elements. This composition obviated the need for external energy sources and allowed for remote manipulation of the tools. Selective enzymatic degradation of biopolymer hinge components triggered closing of the grippers; subsequent reopening was achieved with an orthogonal enzyme. We highlight the utility of these enzymatically triggered tools by demonstrating the biopsy of liver tissue from a model organ system and gripping and releasing an alginate bead. This strategy suggests an approach for the development of smart materials and devices that autonomously reconfigure in response to extremely specific biological environments.