Molecular Biology of the Cell最新文献_第9页

Optimization of the fluorogen-activating protein tag for quantitative protein trafficking and colocalization studies in S. cerevisiae. 优化荧光激活蛋白标签，用于 S. cerevisiae 蛋白定量迁移和共定位研究。

IF 3.1 3区生物学

Molecular Biology of the Cell Pub Date : 2024-07-01 Epub Date: 2024-05-29 DOI: 10.1091/mbc.E24-04-0174

Katherine G Oppenheimer, Natalie A Hager, Ceara K McAtee, Elif Filiztekin, Chaowei Shang, Justina A Warnick, Marcel P Bruchez, Jeffrey L Brodsky, Derek C Prosser, Adam V Kwiatkowski, Allyson F O'Donnell

{"title":"Optimization of the fluorogen-activating protein tag for quantitative protein trafficking and colocalization studies in S. cerevisiae.","authors":"Katherine G Oppenheimer, Natalie A Hager, Ceara K McAtee, Elif Filiztekin, Chaowei Shang, Justina A Warnick, Marcel P Bruchez, Jeffrey L Brodsky, Derek C Prosser, Adam V Kwiatkowski, Allyson F O'Donnell","doi":"10.1091/mbc.E24-04-0174","DOIUrl":"10.1091/mbc.E24-04-0174","url":null,"abstract":"Spatial and temporal tracking of fluorescent proteins (FPs) in live cells permits visualization of proteome remodeling in response to extracellular cues. Historically, protein dynamics during trafficking have been visualized using constitutively active FPs fused to proteins of interest. While powerful, such FPs label all cellular pools of a protein, potentially masking the dynamics of select subpopulations. To help study protein subpopulations, bioconjugate tags, including the fluorogen activation proteins (FAPs), were developed. FAPs are comprised of two components: a single-chain antibody (SCA) fused to the protein of interest and a malachite-green (MG) derivative, which fluoresces only when bound to the SCA. Importantly, the MG derivatives can be either cell-permeant or -impermeant, thus permitting isolated detection of SCA-tagged proteins at the cell surface and facilitating quantitative endocytic measures. To expand FAP use in yeast, we optimized the SCA for yeast expression, created FAP-tagging plasmids, and generated FAP-tagged organelle markers. To demonstrate FAP efficacy, we coupled the SCA to the yeast G-protein coupled receptor Ste3. We measured Ste3 endocytic dynamics in response to pheromone and characterized cis- and trans-acting regulators of Ste3. Our work significantly expands FAP technology for varied applications in S. cerevisiae.","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":null,"pages":null},"PeriodicalIF":3.1,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11244157/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141161980","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Cell type- and transcription-independent spatial proximity between enhancers and promoters. 增强子和启动子之间与细胞类型和转录无关的空间接近性

IF 3.1 3区生物学

Molecular Biology of the Cell Pub Date : 2024-07-01 Epub Date: 2024-05-08 DOI: 10.1091/mbc.E24-02-0082

Yasmine Mian, Li Wang, Adib Keikhosravi, Konnie Guo, Tom Misteli, H Efsun Arda, Elizabeth H Finn

{"title":"Cell type- and transcription-independent spatial proximity between enhancers and promoters.","authors":"Yasmine Mian, Li Wang, Adib Keikhosravi, Konnie Guo, Tom Misteli, H Efsun Arda, Elizabeth H Finn","doi":"10.1091/mbc.E24-02-0082","DOIUrl":"10.1091/mbc.E24-02-0082","url":null,"abstract":"Cell type-specific enhancers are critically important for lineage specification. The mechanisms that determine cell-type specificity of enhancer activity, however, are not fully understood. Most current models for how enhancers function invoke physical proximity between enhancer elements and their target genes. Here, we use an imaging-based approach to examine the spatial relationship of cell type-specific enhancers and their target genes with single-cell resolution. Using high-throughput microscopy, we measure the spatial distance from target promoters to their cell type-specific active and inactive enhancers in individual pancreatic cells derived from distinct lineages. We find increased proximity of all promoter-enhancer pairs relative to non-enhancer pairs separated by similar genomic distances. Strikingly, spatial proximity between enhancers and target genes was unrelated to tissue-specific enhancer activity. Furthermore, promoter-enhancer proximity did not correlate with the expression status of target genes. Our results suggest that promoter-enhancer pairs exist in a distinctive chromatin environment but that genome folding is not a universal driver of cell-type specificity in enhancer function.","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":null,"pages":null},"PeriodicalIF":3.1,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11244156/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140876784","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A PACAP-activated network for secretion requires coordination of Ca²⁺ influx and Ca²⁺ mobilization. PACAP 激活的分泌网络需要协调 Ca2+ 的流入和 Ca2+ 的动员。

IF 3.1 3区生物学

Molecular Biology of the Cell Pub Date : 2024-07-01 Epub Date: 2024-05-17 DOI: 10.1091/mbc.E24-02-0083

Xiaohuan Chen, Nicole A Bell, Breanna L Coffman, Agustin A Rabino, Rafael Garcia-Mata, Paul J Kammermeier, David I Yule, Daniel Axelrod, Alan V Smrcka, David R Giovannucci, Arun Anantharam

{"title":"A PACAP-activated network for secretion requires coordination of Ca2+ influx and Ca2+ mobilization.","authors":"Xiaohuan Chen, Nicole A Bell, Breanna L Coffman, Agustin A Rabino, Rafael Garcia-Mata, Paul J Kammermeier, David I Yule, Daniel Axelrod, Alan V Smrcka, David R Giovannucci, Arun Anantharam","doi":"10.1091/mbc.E24-02-0083","DOIUrl":"10.1091/mbc.E24-02-0083","url":null,"abstract":"Chromaffin cells of the adrenal medulla transduce sympathetic nerve activity into stress hormone secretion. The two neurotransmitters principally responsible for coupling cell stimulation to secretion are acetylcholine and pituitary adenylate activating polypeptide (PACAP). In contrast to acetylcholine, PACAP evokes a persistent secretory response from chromaffin cells. However, the mechanisms by which PACAP acts are poorly understood. Here, it is shown that PACAP induces sustained increases in cytosolic Ca2+ which are disrupted when Ca2+ influx through L-type channels is blocked or internal Ca2+ stores are depleted. PACAP liberates stored Ca2+ via inositol trisphosphate receptors (IP3Rs) on the endoplasmic reticulum (ER), thereby functionally coupling Ca2+ mobilization to Ca2+ influx and supporting Ca2+-induced Ca2+-release. These Ca2+ influx and mobilization pathways are unified by an absolute dependence on phospholipase C epsilon (PLCε) activity. Thus, the persistent secretory response that is a defining feature of PACAP activity, in situ, is regulated by a signaling network that promotes sustained elevations in intracellular Ca2+ through multiple pathways.","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":null,"pages":null},"PeriodicalIF":3.1,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11244167/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140957667","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Differential reliance of CTD-nuclear envelope phosphatase 1 on its regulatory subunit in ER lipid synthesis and storage. CTD 核包膜磷酸酶 1 在 ER 脂质合成和储存过程中对其调节亚基的不同依赖性。

IF 3.1 3区生物学

Molecular Biology of the Cell Pub Date : 2024-07-01 Epub Date: 2024-05-22 DOI: 10.1091/mbc.E23-09-0382

Jake W Carrasquillo Rodríguez, Onyedikachi Uche, Shujuan Gao, Shoken Lee, Michael V Airola, Shirin Bahmanyar

{"title":"Differential reliance of CTD-nuclear envelope phosphatase 1 on its regulatory subunit in ER lipid synthesis and storage.","authors":"Jake W Carrasquillo Rodríguez, Onyedikachi Uche, Shujuan Gao, Shoken Lee, Michael V Airola, Shirin Bahmanyar","doi":"10.1091/mbc.E23-09-0382","DOIUrl":"10.1091/mbc.E23-09-0382","url":null,"abstract":"Lipin 1 is an ER enzyme that produces diacylglycerol, the lipid intermediate that feeds into the synthesis of glycerophospholipids for membrane expansion or triacylglycerol for storage into lipid droplets. CTD-Nuclear Envelope Phosphatase 1 (CTDNEP1) regulates lipin 1 to restrict ER membrane synthesis, but a role for CTDNEP1 in lipid storage in mammalian cells is not known. Furthermore, how NEP1R1, the regulatory subunit of CTDNEP1, contributes to these functions in mammalian cells is not fully understood. Here, we show that CTDNEP1 is reliant on NEP1R1 for its stability and function in limiting ER expansion. CTDNEP1 contains an amphipathic helix at its N-terminus that targets to the ER, nuclear envelope and lipid droplets. We identify key residues at the binding interface of CTDNEP1 and NEP1R1 and show that they facilitate complex formation in vivo and in vitro. We demonstrate that NEP1R1 binding to CTDNEP1 shields CTDNEP1 from proteasomal degradation to regulate lipin 1 and restrict ER size. Unexpectedly, NEP1R1 was not required for CTDNEP1's role in restricting lipid droplet biogenesis. Thus, the reliance of CTDNEP1 function on NEP1R1 depends on cellular demands for membrane production versus lipid storage. Together, our work provides a framework into understanding how the ER regulates lipid synthesis under different metabolic conditions.","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":null,"pages":null},"PeriodicalIF":3.1,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11244170/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141076252","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

From pipette to palette: Bridging science and art at MBoC. 从移液管到调色板：在 MBoC 搭建科学与艺术的桥梁。

IF 3.1 3区生物学

Molecular Biology of the Cell Pub Date : 2024-06-01 DOI: 10.1091/mbc.E24-04-0149

Barry Whittaker, Eric Zeigler, Rafael Garcia-Mata, Jaqulin N Wallace, Jennifer R Morgan, Avital A Rodal, Matthew D Welch

引用次数: 0

Importin α/β mediates nuclear import of individual SUMO E1 subunits and of the holo-enzyme. 导入蛋白α/β介导单个SUMO E1亚基和全酶的核导入。

IF 3.1 3区生物学

Molecular Biology of the Cell Pub Date : 2024-06-01 DOI: 10.1091/mbc.E10-05-0461_cor

引用次数: 0

Drosophila Nhe2 Over-expression Induces Autophagic Cell Death 果蝇 Nhe2 过度表达诱导自噬细胞死亡

IF 3.3 3区生物学

Molecular Biology of the Cell Pub Date : 2024-05-02 DOI: 10.1091/mbc.e24-02-0058

Jobelle Peralta, Blake DuPriest, Daniel Orozco, Juan Reyna Pacheco, Laura Martins, Rachel Ann Soriano, Alan Wong, Ramy Wong, Bree Grillo-Hill

{"title":"Drosophila Nhe2 Over-expression Induces Autophagic Cell Death","authors":"Jobelle Peralta, Blake DuPriest, Daniel Orozco, Juan Reyna Pacheco, Laura Martins, Rachel Ann Soriano, Alan Wong, Ramy Wong, Bree Grillo-Hill","doi":"10.1091/mbc.e24-02-0058","DOIUrl":"https://doi.org/10.1091/mbc.e24-02-0058","url":null,"abstract":"Autophagy is a conserved catabolic process where double membrane-bound structures form around macromolecules or organelles targeted for degradation. Autophagosomes fuse with lysosomes to facilitate degradation and macromolecule recycling for homeostasis or growth in a cell autonomous fashion. In cancer cells, autophagy is often upregulated and helps cancer cells survive nutrient deprivation and stressful growth conditions. Here, we propose that the increased intracellular pH (pHi) common to cancer cells is sufficient to induce autophagic cell death. We previously developed tools to increase pHi in the Drosophila eye via over-expression of DNhe2, resulting in aberrant patterning and reduced tissue size. We examined fly eyes at earlier stages of development and found fewer interommatidial cells. We next tested whether this decrease in cell number was due to increased cell death. We found that the DNhe2-induced cell death was caspase-independent, which is inconsistent with apoptosis. However, this cell death required autophagy genes, which supports autophagy as the mode of cell death. We also found that expression of molecular markers supports increased autophagy. Together, our findings suggest new roles for ion transport proteins in regulating conserved, critical developmental processes and provide evidence for new paradigms in growth control.","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140830293","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Bridge-like lipid transfer protein family member 2 suppresses ciliogenesis. 桥状脂质转移蛋白家族成员2抑制纤毛的生成

IF 3.1 3区生物学

Molecular Biology of the Cell Pub Date : 2024-05-01 Epub Date: 2024-03-27 DOI: 10.1091/mbc.E24-02-0065

Jan Parolek, Christopher G Burd

{"title":"Bridge-like lipid transfer protein family member 2 suppresses ciliogenesis.","authors":"Jan Parolek, Christopher G Burd","doi":"10.1091/mbc.E24-02-0065","DOIUrl":"10.1091/mbc.E24-02-0065","url":null,"abstract":"Bridge-like lipid transfer protein family member 2 (BLTP2) is an evolutionary conserved protein with unknown function(s). The absence of BLTP2 in Drosophila melanogaster results in impaired cellular secretion and larval death, while in mice (Mus musculus), it causes preweaning lethality. Structural predictions propose that BLTP2 belongs to the repeating β-groove domain-containing (also called the VPS13) protein family, forming a long tube with a hydrophobic core, suggesting that it operates as a lipid transfer protein (LTP). We establish BLTP2 as a negative regulator of ciliogenesis in RPE-1 cells based on a strong genetic interaction with WDR44, a gene that also suppresses ciliogenesis. Like WDR44, BLTP2 localizes to membrane contact sites involving the endoplasmic reticulum and the tubular endosome network in HeLa cells and that BLTP2 depletion enhanced ciliogenesis in RPE-1 cells grown in serum-containing medium, a condition where ciliogenesis is normally suppressed. This study establishes human BLTP2 as a putative LTP acting between tubular endosomes and ER that regulates primary cilium biogenesis.","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":null,"pages":null},"PeriodicalIF":3.1,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11151097/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140306126","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Sec18 binds the tethering/SM complex HOPS to engage the Qc-SNARE for membrane fusion. Sec18 与系留/SM 复合物 HOPS 结合，使 Qc-SNARE 参与膜融合。

IF 3.1 3区生物学

Molecular Biology of the Cell Pub Date : 2024-05-01 Epub Date: 2024-03-27 DOI: 10.1091/mbc.E24-02-0060

Amy Orr, William Wickner

{"title":"Sec18 binds the tethering/SM complex HOPS to engage the Qc-SNARE for membrane fusion.","authors":"Amy Orr, William Wickner","doi":"10.1091/mbc.E24-02-0060","DOIUrl":"10.1091/mbc.E24-02-0060","url":null,"abstract":"Membrane fusion is regulated by Rab GTPases, their tethering effectors such as HOPS, SNARE proteins on each fusion partner, SM proteins to catalyze SNARE assembly, Sec17 (SNAP), and Sec18 (NSF). Though concentrated HOPS can support fusion without Sec18, we now report that fusion falls off sharply at lower HOPS levels, where direct Sec18 binding to HOPS restores fusion. This Sec18-dependent fusion needs adenine nucleotide but neither ATP hydrolysis nor Sec17. Sec18 enhances HOPS recognition of the Qc-SNARE. With high levels of HOPS, Qc has a Km for fusion of a few nM. Either lower HOPS levels, or substitution of a synthetic tether for HOPS, strikingly increases the Km for Qc to several hundred nM. With dilute HOPS, Sec18 returns the Km for Qc to low nM. In contrast, HOPS concentration and Sec18 have no effect on Qb-SNARE recognition. Just as Qc is required for fusion but not for the initial assembly of SNAREs in trans, impaired Qc recognition by limiting HOPS without Sec18 still allows substantial trans-SNARE assembly. Thus, in addition to the known Sec18 functions of disassembling SNARE complexes, oligomerizing Sec17 for membrane association, and allowing Sec17 to drive fusion without complete SNARE zippering, we report a fourth Sec18 function, the Sec17-independent binding of Sec18 to HOPS to enhance functional Qc-SNARE engagement.","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":null,"pages":null},"PeriodicalIF":3.1,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11151092/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140306128","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Drosophila Fog/Cta and T48 pathways have overlapping and distinct contributions to mesoderm invagination. 果蝇的 Fog/Cta 和 T48 通路对中胚层内陷有重叠和不同的贡献。

IF 3.1 3区生物学

Molecular Biology of the Cell Pub Date : 2024-05-01 Epub Date: 2024-03-27 DOI: 10.1091/mbc.E24-02-0050

Uzuki Horo, D Nathaniel Clarke, Adam C Martin

{"title":"Drosophila Fog/Cta and T48 pathways have overlapping and distinct contributions to mesoderm invagination.","authors":"Uzuki Horo, D Nathaniel Clarke, Adam C Martin","doi":"10.1091/mbc.E24-02-0050","DOIUrl":"10.1091/mbc.E24-02-0050","url":null,"abstract":"The regulation of the cytoskeleton by multiple signaling pathways, sometimes in parallel, is a common principle of morphogenesis. A classic example of regulation by parallel pathways is Drosophila gastrulation, where the inputs from the Folded gastrulation (Fog)/Concertina (Cta) and the T48 pathways induce apical constriction and mesoderm invagination. Whether there are distinct roles for these separate pathways in regulating the complex spatial and temporal patterns of cytoskeletal activity that accompany early embryo development is still poorly understood. We investigated the roles of the Fog/Cta and T48 pathways and found that, by themselves, the Cta and T48 pathways both promote timely mesoderm invagination and apical myosin II accumulation, with Cta being required for timely cell shape change ahead of mitotic cell division. We also identified distinct functions of T48 and Cta in regulating cellularization and the uniformity of the apical myosin II network, respectively. Our results demonstrate that both redundant and distinct functions for the Fog/Cta and T48 pathways exist.","PeriodicalId":18735,"journal":{"name":"Molecular Biology of the Cell","volume":null,"pages":null},"PeriodicalIF":3.1,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11151099/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140306213","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0