The Golgi is a dynamic organelle whose correct assembly is crucial for cellular homeostasis. Perturbations in Golgi structure are associated with numerous disorders from neurodegeneration to cancer. ...However, whether and how dispersal of the Golgi apparatus is actively regulated under stress, and the consequences of Golgi dispersal, remain unknown. Here we demonstrate that 26S proteasomes are associated with the cytosolic surface of Golgi membranes to facilitate Golgi Apparatus-Related Degradation (GARD) and degradation of GM130 in response to Golgi stress. The degradation of GM130 is dependent on p97/VCP and 26S proteasomes, and required for Golgi dispersal. Finally, we show that perturbation of Golgi homeostasis induces cell death of multiple myeloma in vitro and in vivo, offering a therapeutic strategy for this malignancy. Taken together, this work reveals a mechanism of Golgi-localized proteasomal degradation, providing a functional link between proteostasis control and Golgi architecture, which may be critical in various secretion-related pathologies.
Pancreatic beta cells have been shown to be heterogeneous at multiple levels. However, spatially interrogating transcriptional heterogeneity in the intact tissue has been challenging. Here, we ...developed an optimized protocol for single-molecule transcript imaging in the intact pancreas and used it to identify a sub-population of “extreme” beta cells with elevated mRNA levels of insulin and other secretory genes. Extreme beta cells contain higher ribosomal and proinsulin content but lower levels of insulin protein in fasted states, suggesting they may be tuned for basal insulin secretion. They exhibit a distinctive intra-cellular polarization pattern, with elevated mRNA concentrations in an apical ER-enriched compartment, distinct from the localization of nascent and mature proteins. The proportion of extreme cells increases in db/db diabetic mice, potentially facilitating the required increase in basal insulin. Our results thus highlight a sub-population of beta cells that may carry distinct functional roles along physiological and pathological timescales.
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•Islets contain a minority of “extreme” beta cells with elevated insulin mRNA levels•Elevated proinsulin yet lower insulin protein suggests these are basal secretors•Extreme cells exhibit discordant apical-basal mRNA and protein localization•The proportion of extreme cells increases in insulin-resistant animals
Using an optimized protocol for single-molecule transcript imaging of the intact pancreas, Farack, Golan, et al. reveal that a small subset of beta cells harbors elevated levels of ribosomes and insulin mRNA, suggesting a sub-specialization in basal insulin secretion.
A novel therapeutic approach in cancer, attempting to stimulate host anti-tumor immunity, involves blocking of immune checkpoints. Lymphocyte activation gene 3 (LAG3) is an immune checkpoint receptor ...expressed on activated/exhausted T cells. When engaged by the major histocompatibility complex (MHC) class II molecules, LAG3 negatively regulates T-cell function, thereby contributing to tumor escape. Intriguingly, a soluble LAG3 variant activates both immune and malignant MHC class II-presenting cells. In the study herein, we examined the role of LAG3 in the pathogenesis of chronic lymphocytic leukemia, an MHC class II-presenting malignancy, and show that chronic lymphocytic leukemia cells express and secrete LAG3. High levels of surface and soluble LAG3 were associated with the unmutated immunoglobulin variable heavy chain leukemic subtype and a shorter median time from diagnosis to first treatment. Utilizing a mechanism mediated through MHC class II engagement, recombinant soluble LAG3-Ig fusion protein, LAG3-Fc, activated chronic lymphocytic leukemia cells, induced anti-apoptotic pathways and protected the cells from spontaneous apoptosis, effects mediated by SYK, BTK and MAPK signaling. Moreover, LAG3 blocking antibody enhanced
T-cell activation. Our data suggest that soluble LAG3 promotes leukemic cell activation and anti-apoptotic effects through its engagement with MHC class II. Furthermore, MHC class II-presenting chronic lymphocytic leukemia cells may affect LAG3-presenting T cells and impose immune exhaustion on their microenvironment; hence, blocking LAG3-MHC class II interactions is a potential therapeutic target in chronic lymphocytic leukemia.
Abstract Mitochondria are maternally inherited, but the mechanisms underlying paternal mitochondrial elimination after fertilization are far less clear. Using Drosophila , we show that special ...egg-derived multivesicular body vesicles promote paternal mitochondrial elimination by activating an LC3-associated phagocytosis-like pathway, a cellular defense pathway commonly employed against invading microbes. Upon fertilization, these egg-derived vesicles form extended vesicular sheaths around the sperm flagellum, promoting degradation of the sperm mitochondrial derivative and plasma membrane. LC3-associated phagocytosis cascade of events, including recruitment of a Rubicon-based class III PI(3)K complex to the flagellum vesicular sheaths, its activation, and consequent recruitment of Atg8/LC3, are all required for paternal mitochondrial elimination. Finally, lysosomes fuse with strings of large vesicles derived from the flagellum vesicular sheaths and contain degrading fragments of the paternal mitochondrial derivative. Given reports showing that in some mammals, the paternal mitochondria are also decorated with Atg8/LC3 and surrounded by multivesicular bodies upon fertilization, our findings suggest that a similar pathway also mediates paternal mitochondrial elimination in other flagellated sperm-producing organisms.
TmaR, the only known pole-localizer protein in Escherichia coli, was shown to cluster at the cell poles and control localization and activity of the major sugar regulator in a tyrosine ...phosphorylation-dependent manner. Here, we show that TmaR assembles by phase separation (PS) via heterotypic interactions with RNA in vivo and in vitro. An unbiased automated mutant screen combined with directed mutagenesis and genetic manipulations uncovered the importance of a predicted nucleic-acid-binding domain, a disordered region, and charged patches, one containing the phosphorylated tyrosine, for TmaR condensation. We demonstrate that, by protecting flagella-related transcripts, TmaR controls flagella production and, thus, cell motility and biofilm formation. These results connect PS in bacteria to survival and provide an explanation for the linkage between metabolism and motility. Intriguingly, a point mutation or increase in its cellular concentration induces irreversible liquid-to-solid transition of TmaR, similar to human disease-causing proteins, which affects cell morphology and division.
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•TmaR undergoes phase separation (PS) via heterotypic interactions with RNA•Combined biased and unbiased mutagenesis revealed the requisites for TmaR PS•TmaR condensates protect flagellar mRNAs, thus controlling flagella formation•TmaR may transition from liquid to solid similar to disease-causing proteins
TmaR is a bacterial pole localizer that regulates sugar metabolism. Szoke et al. show that TmaR clusters are formed by phase separation. By protecting flagella-related transcripts, TmaR controls flagella biogenesis and motility. Their results show that phase separation is important for bacterial survival and explain the linkage between metabolism and motility.
Mono-allelic germline disruptions of the transcription factor GATA2 result in a propensity for developing myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), affecting more than 85% of ...carriers. How a partial loss of GATA2 functionality enables leukemic transformation years later is unclear. This question has remained unsolved mainly due to the lack of informative models, as
Gata2
heterozygote mice do not develop hematologic malignancies. Here we show that two different germline
Gata2
mutations (Tg
Erg/Gata2
het
and Tg
Erg/Gata2
L359V
) accelerate AML in mice expressing the human hematopoietic stem cell regulator ERG. Analysis of
Erg/Gata2
het
fetal liver and bone marrow-derived hematopoietic cells revealed a distinct pre-leukemic phenotype. This was characterized by enhanced transition from stem to progenitor state, increased proliferation, and a striking mitochondrial phenotype, consisting of highly expressed oxidative-phosphorylation-related gene sets, elevated oxygen consumption rates, and notably, markedly distorted mitochondrial morphology. Importantly, the same mitochondrial gene-expression signature was observed in human AML harboring
GATA2
aberrations. Similar to the observations in mice, non-leukemic bone marrows from children with germline
GATA2
mutation demonstrated marked mitochondrial abnormalities. Thus, we observed the tumor suppressive effects of GATA2 in two germline
Gata2
genetic mouse models. As oncogenic mutations often accumulate with age, GATA2 deficiency-mediated priming of hematopoietic cells for oncogenic transformation may explain the earlier occurrence of MDS/AML in patients with
GATA2
germline mutation. The mitochondrial phenotype is a potential therapeutic opportunity for the prevention of leukemic transformation in these patients.
I In the last decade, the B-cell receptor has emerged as a pivotal stimulus in the pathogenesis of chronic lymphocytic leukemia, and a very feasible therapeutic target in this disease. B-cell ...receptor responsiveness in chronic lymphocytic leukemia cells is heterogeneous among patients and correlates with aggressiveness of the disease. Here we show, for the first time, that SLP76, a key scaffold protein in T-cell receptor signaling, is ectopically expressed in chronic lymphocytic leukemia cells, with variable levels among patients, and correlates positively with unmutated immunoglobulin heavy chain variable gene status and ZAP-70 expression. We found that SLP76 was functionally active in chronic lymphocytic leukemia cells. A SYK-dependent basal level of phosphorylated SLP76 exists in the cells, and upon B-cell receptor engagement, SLP76 tyrosine phosphorylation is significantly enhanced concomitantly with increased physical association with BTK. B-cell receptor-induced SLP76 phosphorylation is mediated by upstream signaling events involving LCK and SYK. Knockdown of SLP76 in the cells resulted in decreased induction of BTK, PLCγ2 and IκB phosphorylation, as well as cell viability after B-cell receptor activation with anti-IgM. Consistent with our biochemical findings, high total SLP76 expression in chronic lymphocytic leukemia cells correlated with a more aggressive disease course.
SLP76 is ectopically expressed in chronic lymphocytic leukemia cells where it plays a role in B-cell receptor signaling.
Spliceosome machinery mutations are common early mutations in myeloid malignancies; however, effective targeted therapies against them are still lacking. In the current study, we used an in vitro ...high-throughput drug screen among four different isogenic cell lines and identified RKI-1447, a Rho-associated protein kinase inhibitor, as selective cytotoxic effector of SRSF2 mutant cells. RKI-1447 targeted SRSF2 mutated primary human samples in xenografts models. RKI-1447 induced mitotic catastrophe and induced major reorganization of the microtubule system and severe nuclear deformation. Transmission electron microscopy and 3D light microscopy revealed that SRSF2 mutations induce deep nuclear indentation and segmentation that are apparently driven by microtubule-rich cytoplasmic intrusions, which are exacerbated by RKI-1447. The severe nuclear deformation in RKI-1447-treated SRSF2 mutant cells prevents cells from completing mitosis. These findings shed new light on the interplay between microtubules and the nucleus and offers new ways for targeting pre-leukemic SRSF2 mutant cells.
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•RKI-1447 identified in high-throughput screening as potent for SRSF2 mutant cells•RKI-1447 effective in xenograft model against primary human leukemia samples•The Inhibitor causes mitotic catastrophe, cytoskeleton reorganization, severe nuclear deformation•The results point to a new therapeutic approach for leukemias involving SRSF2 mutations
Molecular biology; Cell biology; Cancer
Controlling the morphology of crystalline materials is challenging, as crystals have a strong tendency toward thermodynamically stable structures. Yet, organisms form crystals with distinct ...morphologies, such as the plate-like guanine crystals produced by many terrestrial and aquatic species for light manipulation. Regulation of crystal morphogenesis was hypothesized to entail physical growth restriction by the surrounding membrane, combined with fine-tuned interactions between organic molecules and the growing crystal. Using cryo-electron tomography of developing zebrafish larvae, we found that guanine crystals form via templated nucleation of thin leaflets on preassembled scaffolds made of 20-nm-thick amyloid fibers. These leaflets then merge and coalesce into a single plate-like crystal. Our findings shed light on the biological regulation of crystal morphogenesis, which determines their optical properties.
Autophagy eliminates cytoplasmic material by engulfment in membranous vesicles targeted for lysosome degradation. Nonselective autophagy coordinates sequestration of bulk cargo with the growth of the ...isolation membrane (IM) in a yet-unknown manner. Here, we show that in the budding yeast Saccharomyces cerevisiae, IMs expand while maintaining a rim sufficiently wide for sequestration of large cargo but tight enough to mature in due time. An obligate complex of Atg24/Snx4 with Atg20 or Snx41 assembles locally at the rim in a spatially extended manner that specifically depends on autophagic PI(3)P. This assembly stabilizes the open rim to promote autophagic sequestration of large cargo in correlation with vesicle expansion. Moreover, constriction of the rim by the PI(3)P-dependent Atg2-Atg18 complex and clearance of PI(3)P by Ymr1 antagonize rim opening to promote autophagic maturation and consumption of small cargo. Tight regulation of membrane rim aperture by PI(3)P thus couples the mechanism and physiology of nonselective autophagy.
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•The nonselective yeast phagophore expands spherically while maintaining a narrow rim•The Atg2-Atg18 complex maintains rim constriction during membrane expansion•Local assembly of an autophagy-specific Atg20-Atg24 stabilizes a widely open rim•Rim stabilization is essential for autophagic sequestration of large cytoplasmic cargo
Autophagosome biogenesis couples sequestration of cargo to expansion of the autophagic membrane in an elusive manner. Shatz et al. show that the rim of the nonselective yeast phagophore is tightly regulated to balance the inclusion of large cytoplasmic cargo with productive maturation in due time.
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