Dataset Information

Systematic spatial mapping of proteins at exocytic and endocytic structures.

ABSTRACT: Vesicular secretion (exocytosis) involves the release and then compensatory recycling of vesicle components through endocytosis. This fundamental cellular process is controlled by the coordinated assembly and interactions of dozens of proteins at the plasma membrane. Understanding the molecular composition of individual exocytic and endocytic structures and their organization across the plasma membrane is critical to understanding the behavior and regulation of these two cellular processes. Here we develop a high-resolution and high-throughput fluorescence imaging-based approach for the unbiased mapping of 78 proteins at single exocytic vesicles and endocytic structures in neuroendocrine PC12 cells. This analysis uses two-color single-frame images to provide a systems-level map of the steady-state distributions of proteins at individual exocytic and endocytic structures in the cell. Along with this quantitative map, we find that both calcium-regulated exocytic vesicles (dense core vesicles) and endocytic structures (clathrin-coated structures) and the proteins associated with these structures exhibit a random spatial distribution in unstimulated neuroendocrine PC12 cells. This approach is broadly applicable for quantitatively mapping the molecular composition and spatial organization of discrete cellular processes with central molecular hubs.

SUBMITTER: Larson BT

PROVIDER: S-EPMC4072581 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Systematic spatial mapping of proteins at exocytic and endocytic structures.

Larson Ben T BT Sochacki Kem A KA Kindem Jonathan M JM Taraska Justin W JW

Molecular biology of the cell 20140507 13

Vesicular secretion (exocytosis) involves the release and then compensatory recycling of vesicle components through endocytosis. This fundamental cellular process is controlled by the coordinated assembly and interactions of dozens of proteins at the plasma membrane. Understanding the molecular composition of individual exocytic and endocytic structures and their organization across the plasma membrane is critical to understanding the behavior and regulation of these two cellular processes. Here ...[more]

PMID: 24807904

Similar Datasets

Project description:Toxoplasma gondii is a widely prevalent parasite of animals and humans that causes a broad spectrum of conditions ranging from asymptomatic or mild cold-like infections to abortion, severe ocular disease, encephalitis, and sometimes death. It is a member of the phylum Apicomplexa that also includes such significant human pathogens as Plasmodium spp. and Cryptosporidium spp. causing malaria and diarrheal disease, respectively. The toxoplasma cell has a multilayered pellicle: under the plasma membrane lies the inner membrane complex (IMC) comprised of flat membrane sacks supported by cortical microtubules and a meshwork of cytoskeletal filaments. The IMC acts as a mechanical barrier for membrane vesicles trafficking to and from the plasma membrane, necessitating the existence of dedicated cites for endo- and exocytosis where the IMC is interrupted. One such site is located at the apical end of the cell where the contents of the apical secretory organelles is exocytosed upon invasion. However, endocytosis in Toxoplasma and other apicomplexans is poorly understood, although conspicuous invaginations of the plasma membrane observed microscopically have been dubbed the micropore and postulated to be involved in endocytosis. We found that the endocytic markers including the subunits α and μ of the AP2 adaptor complex, dynamin-related protein C (DrpC), an Eps15 homology domain-containing protein, and the kelch-domain protein K13 localise to distinct persistent foci in the pellicle of Toxoplasma. Another type of persistent punctate structures found in the IMC are the enigmatic apical annuli whose function is completely unknown. We have discovered a new component of the annuli which is a putative LMBR1 family region protein embedded into the plasma membrane. Our experiments indicate that this protein – and annuli by extension – has a role in exocytosis. To better understand the molecular composition of these endocytic and exocytic structures and gain an insight into their function, we genetically fused them with a promiscuous bacterial biotin-ligase BirA* and used them as baits in proximity-dependent biotin identification experiments (BioID). We employed TMT10plex labelling and MS3-level tandem mass spectrometry with synchronous precursor selection (SPS-MS3) to achieve accurate quantification of multiple samples. Several hundred proteins have been identified and quantified in three biological replicates for every bait and the parental cell line control. Proteins significantly enriched in the BirA*-tagged cells relative to the control have been identified using linear modelling with LIMMA.

Project description:Background Approaches in malaria risk mapping continue to advance in scope with the advent of geostatistical techniques spanning both the spatial and temporal domains. A substantive review of the merits of the methods and covariates used to map malaria risk has not been undertaken. Therefore, this review aimed to systematically retrieve, summarise methods and examine covariates that have been used for mapping malaria risk in sub-Saharan Africa (SSA). Methods A systematic search of malaria risk mapping studies was conducted using PubMed, EBSCOhost, Web of Science and Scopus databases. The search was restricted to refereed studies published in English from January 1968 to April 2020. To ensure completeness, a manual search through the reference lists of selected studies was also undertaken. Two independent reviewers completed each of the review phases namely: identification of relevant studies based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, data extraction and methodological quality assessment using a validated scoring criterion. Results One hundred and seven studies met the inclusion criteria. The median quality score across studies was 12/16 (range: 7–16). Approximately half (44%) of the studies employed variable selection techniques prior to mapping with rainfall and temperature selected in over 50% of the studies. Malaria incidence (47%) and prevalence (35%) were the most commonly mapped outcomes, with Bayesian geostatistical models often (31%) the preferred approach to risk mapping. Additionally, 29% of the studies employed various spatial clustering methods to explore the geographical variation of malaria patterns, with Kulldorf scan statistic being the most common. Model validation was specified in 53 (50%) studies, with partitioning data into training and validation sets being the common approach. Conclusions Our review highlights the methodological diversity prominent in malaria risk mapping across SSA. To ensure reproducibility and quality science, best practices and transparent approaches should be adopted when selecting the statistical framework and covariates for malaria risk mapping. Findings underscore the need to periodically assess methods and covariates used in malaria risk mapping; to accommodate changes in data availability, data quality and innovation in statistical methodology.

Dataset Information

Systematic spatial mapping of proteins at exocytic and endocytic structures.

Publications

Systematic spatial mapping of proteins at exocytic and endocytic structures.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets