The HEAT repeat protein HPO-27 is a lysosome fission factor.

Lysosomes are degradation and signalling centres crucial for homeostasis, development and ageing1. To meet diverse cellular demands, lysosomes remodel their morphology and function through constant fusion and fission2,3. Little is known about the molecular basis of fission. Here we identify HPO-27, a conserved HEAT repeat protein, as a lysosome scission factor in Caenorhabditis elegans. Loss of HPO-27 impairs lysosome fission and leads to an excessive tubular network that ultimately collapses. HPO-27 and its human homologue MROH1 are recruited to lysosomes by RAB-7 and enriched at scission sites. Super-resolution imaging, negative-staining electron microscopy and in vitro reconstitution assays reveal that HPO-27 and MROH1 self-assemble to mediate the constriction and scission of lysosomal tubules in worms and mammalian cells, respectively, and assemble to sever supported membrane tubes in vitro. Loss of HPO-27 affects lysosomal morphology, integrity and degradation activity, which impairs animal development and longevity. Thus, HPO-27 and MROH1 act as self-assembling scission factors to maintain lysosomal homeostasis and function.

RBM15 condensates modulate m6A modification of STYK1 to promote tumorigenesis.

RBM15 expression is recurrently upregulated in several types of malignant tissues, and its high expression level is typically associated with poor prognosis. However, whether and how RBM15 is involved in the tumor progression remains unclear. In this study, we found that overexpressing RBM15 in NIH3T3 cells was able to enhance proliferation rate in vitro and induced subcutaneous tumor formation in vivo. Moreover, we imaged the subcellular localization of RBM15 with our home-built structured illumination super-resolution microscopy, and revealed that RBM15 formed substantial condensates dispersed in the nucleus, undergoing dynamic fusion and fission activities. These condensates were partially colocalized with m6A-modified transcripts in the nucleus. In addition, we confirmed that RBM15 formed "liquid-like" droplets in a protein/salt concentration-dependent manner in vitro, and the addition of RNA further enhanced its phase-separation propensity. To identify downstream targets of RBM15, we performed meRIP-seq and RNA-seq, revealing that RBM15 preferentially bound to and promoted the m6A modification on the mRNA of Serine/threonine/tyrosine kinase 1 (STYK1), thereby enhancing its stability. The upregulated STYK1 expression caused MAPK hyperactivation, thereby leading to oncogenic transformation of NIH3T3 cells.

Vam6 reduces iNKT cell function in tumor via modulating AMPK/mTOR pathways.

Activation of mTORC1 is essential for anti-tumor function of iNKT cells. The mechanisms underlying impaired mTORC1 activation in intratumoral iNKT cells remain unclear. Via generating Vam6+/- mice and using flow cytometry, image approach, and RNA sequencing, we studied the role of Vam6 in controlling mTORC1 activation and intratumoral iNKT cell functions. Here, we find that increased Vam6 expression in intratumoral iNKT cells leads to impaired mTORC1 activation and IFN-γ production. Mechanistically, Vam6 in iNKT cells is essential for Rab7a-Vam6-AMPK complex formation and thus for recruitment of AMPK to lysosome to activate AMPK, a negative regulator of mTORC1. Additionally, Vam6 relieves inhibitory effect of VDAC1 on Rab7a-Vam6-AMPK complex formation at mitochondria-lysosome contact site. Moreover, we report that lactic acid produced by tumor cells increases Vam6 expression in iNKT cells. Given the key roles of increased Vam6 in promoting AMPK activation in intratumoral iNKT cells, reducing Vam6 expression signifificantly enhances the mTORC1 activation in intratumoral iNKT cells as well as their anti-tumor effificacy. Together, we propose Vam6 as a target for iNKT cell-based immunotherapy.

Circulating exosomal microRNA-18a-5p accentuates intestinal inflammation in Hirschsprung-associated enterocolitis by targeting RORA.

The relevance of stem cell-derived exosomes has been implicated in necrotizing enterocolitis, while the involvement of serum-derived exosomes from children with Hirschsprung-associated enterocolitis (HAEC) in pathogenesis of HAEC remains unclear. This study set to identify the roles of exosomal microRNA (miR)-18a-5p from sera of HAEC patients in human-derived colonic epithelial NCM460 cells and in mice with HAEC. Exosomes were isolated from the sera of healthy children (Healthy-exo), patients with Hirschsprung's disease (HSCR) (HSCR-exo) or HAEC (HAEC-exo). A microarray analysis of miRNAs was implemented to assess the enrichment of miRNAs in these exosomes. HAEC-exo was significantly enriched in miR-18a-5p. HAEC-exo led to the generation of a pro-inflammatory microenvironment, inhibition of cellular DNA synthesis, and promotion of apoptosis in NCM460 cells. Mechanistically, miR-18a-5p targeted and repressed retinoid-related orphan receptor α (RORA) expression, thereby regulating the Sirtuin 1 (SIRT1)/nuclear factor-kappa B (NFκB) pathway. Overexpression of RORA ameliorated inflammatory damage in NCM460 cells caused by exosomal miR-18a-5p. HAEC-exo exacerbated inflammatory damage in HAEC mice, and this facilitation was reversed after RORA overexpression. Collectively, exosomal miR-18a-5p was a promoter of HAEC, which induces the intestine cell apoptosis and inflammatory responses through the inhibition of SIRT1/NFκB pathway by targeting RORA.

N6-methyladenosine modification of MALAT1 promotes metastasis via reshaping nuclear speckles.

N6-methyladenosine (m6A), one of the most prevalent RNA post-transcriptional modifications, is involved in numerous biological processes. In previous studies, the functions of m6A were typically identified by perturbing the activity of the methyltransferase complex. Here, we dissect the contribution of m6A to an individual-long noncoding RNA-metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). The mutant MALAT1 lacking m6A-motifs significantly suppressed the metastatic potential of cancer cells both in vitro and in vivo in mouse. Super-resolution imaging showed that the concatenated m6A residues on MALAT1 acted as a scaffold for recruiting YTH-domain-containing protein 1 (YTHDC1) to nuclear speckles. We further reveal that the recognition of MALAT1-m6A by YTHDC1 played a critical role in maintaining the composition and genomic binding sites of nuclear speckles, which regulate the expression of several key oncogenes. Furthermore, artificially tethering YTHDC1 onto m6A-deficient MALAT1 largely rescues the metastatic potential of cancer cells.

Instant multicolor super-resolution microscopy with deep convolutional neural network.

Multicolor super-resolution (SR) microscopy plays a critical role in cell biology research and can visualize the interactions between different organelles and the cytoskeleton within a single cell. However, more color channels bring about a heavier budget for imaging and sample preparation, and the use of fluorescent dyes of higher emission wavelengths leads to a worse spatial resolution. Recently, deep convolutional neural networks (CNNs) have shown a compelling capability in cell segmentation, super-resolution reconstruction, image restoration, and many other aspects. Taking advantage of CNN's strong representational ability, we devised a deep CNN-based instant multicolor super-resolution imaging method termed IMC-SR and demonstrated that it could be used to separate different biological components labeled with the same fluorophore, and generate multicolor images from a single super-resolution image in silico. By IMC-SR, we achieved fast three-color live-cell super-resolution imaging with ~100 nm resolution over a long temporal duration, revealing the complicated interactions between multiple organelles and the cytoskeleton in a single COS-7 cell.