bims-momema 2022-03-27 papers

Issue of 2022‒03‒27
four papers selected by
Harilaos Filippakis
Harvard University

Methods Mol Biol. 2022 ;2442 391-411

Evaluating the Role of Galectins in Clathrin-Independent Endocytosis.

Mohit P Mathew, Julie G Donaldson, John A Hanover.

Galectin-3 is a chimeric galectin involved in diverse intracellular and extracellular functions. Galectin-3 is synthesized in the cytoplasm and then released extracellularly by a poorly understood non-canonical secretion mechanism. As a result, it can play important roles both inside and outside the cell. One important extracellular role of galectin-3 is in modulating clathrin-independent endocytosis (CIE), a form of cellular internalization that is still not well understood. CIE, unlike clathrin-mediated endocytosis, has neither defined signaling sequences nor cytoplasmic machinery. As a result, extracellular interactions like the galectin-glycan interactions are thought to directly drive changes in CIE. This chapter discusses the methods designed to study the role of galectin-glycan interactions in CIE, which have provided us with insight into the functions of galectin-3 and cell surface glycans during CIE cargo internalization. These methods include media supplementation for metabolic glycoengineering, antibody internalization assays, lectin panels to assay changes in glycan patterns, exogenous galectin-3 supplementation, galectin-3 secretion assays, and in vitro assays to monitor the effect of galectins on CIE.

Keywords: Antibody internalization assay; Clathrin-independent endocytosis; Galectin-3; Galectin-3 secretion assay

DOI: https://doi.org/10.1007/978-1-0716-2055-7_21

Biomolecules. 2022 Mar 02. pii: 387. [Epub ahead of print]12(3):

Amino Acid Signaling for TOR in Eukaryotes: Sensors, Transducers, and a Sustainable Agricultural fuTORe.

Nanticha Lutt, Jacob O Brunkard.

Eukaryotic cells monitor and regulate metabolism through the atypical protein kinase target of rapamycin (TOR) regulatory hub. TOR is activated by amino acids in animals and fungi through molecular signaling pathways that have been extensively defined in the past ten years. Very recently, several studies revealed that TOR is also acutely responsive to amino acid metabolism in plants, but the mechanisms of amino acid sensing are not yet established. In this review, we summarize these discoveries, emphasizing the diversity of amino acid sensors in human cells and highlighting pathways that are indirectly sensitive to amino acids, i.e., how TOR monitors changes in amino acid availability without a bona fide amino acid sensor. We then discuss the relevance of these model discoveries to plant biology. As plants can synthesize all proteinogenic amino acids from inorganic precursors, we focus on the possibility that TOR senses both organic metabolites and inorganic nutrients. We conclude that an evolutionary perspective on nutrient sensing by TOR benefits both agricultural and biomedical science, contributing to ongoing efforts to generate crops for a sustainable agricultural future.

Keywords: Arabidopsis thaliana; Castor1; GATOR; GCN2; Ragulator; Sestrin2; amino acid signaling; mTOR; metabolism; target of rapamycin

DOI: https://doi.org/10.3390/biom12030387

Microorganisms. 2022 Mar 03. pii: 552. [Epub ahead of print]10(3):

Internalization of Polymeric Bacterial Peptidoglycan Occurs through Either Actin or Dynamin Dependent Pathways.

Narcis I Popescu, Jackie Cochran, Elizabeth Duggan, Jędrzej Kluza, Robert Silasi, Kenneth Mark Coggeshall.

Peptidoglycan (PGN), a polymeric glycan macromolecule, is a major constituent of the bacterial cell wall and a conserved pathogen-associated molecular pattern (PAMP) that triggers immune responses through cytosolic sensors. Immune cells encounter both PGN polymers and hydrolyzed muropeptides during infections, and primary human innate immune cells respond better to polymeric PGN than the minimal bioactive subunit muramyl dipeptide (MDP). While MDP is internalized through macropinocytosis and/or clathrin-mediated endocytosis, the internalization of particulate polymeric PGN is unresolved. We show here that PGN macromolecules isolated from Bacillus anthracis display a broad range of sizes, making them amenable for multiple internalization pathways. Pharmacologic inhibition indicates that PGN primarily, but not exclusively, is internalized by actin-dependent endocytosis. An alternate clathrin-independent but dynamin dependent pathway supports 20-30% of PGN uptake. In primary monocytes, this alternate pathway does not require activities of RhoA, Cdc42 or Arf6 small GTPases. Selective inhibition of PGN uptake shows that phagolysosomal trafficking, processing and downstream immune responses are drastically affected by actin depolymerization, while dynamin inhibition has a smaller effect. Overall, we show that polymeric PGN internalization occurs through two endocytic pathways with distinct potentials to trigger immune responses.

Keywords: CIE; bacteria; internalization; monocytes; peptidoglycan

DOI: https://doi.org/10.3390/microorganisms10030552

Viruses. 2022 Feb 28. pii: 496. [Epub ahead of print]14(3):

Porcine Deltacoronavirus (PDCoV) Entry into PK-15 Cells by Caveolae-Mediated Endocytosis.

Shiqian Li, Dai Xiao, Yujia Zhao, Luwen Zhang, Rui Chen, Weizhe Liu, Yimin Wen, Yijie Liao, Yiping Wen, Rui Wu, Xinfeng Han, Qin Zhao, Senyan Du, Qigui Yan, Xintian Wen, Sanjie Cao, Xiaobo Huang.

(1) Background: Porcine deltacoronavirus (PDCoV) is a newly emerged enteric virus affecting pig breeding industries worldwide, and its pathogenic mechanism remains unclear. (2) Methods: In this study, we preliminarily identified the endocytic pathway of PDCoV in PK-15 cells, using six chemical inhibitors (targeting clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis pathway and endosomal acidification), overexpression of dominant-negative (DN) mutants to treat PK-15 cells and proteins knockdown. (3) Results: The results revealed that PDCoV entry was not affected after treatment with chlorpromazine (CPZ), 5-(N-ethyl-N-isopropyl) amiloride (EIPA)or ammonium chloride (NH4Cl), indicating that the entry of PDCoV into PK-15 cells were clathrin-, micropinocytosis-, PH-independent endocytosis. Conversely, PDCoV infection was sensitive to nystatin, dynasore and methyl-β-cyclodextrin (MβCD) with reduced PDCoV internalization, indicating that entry of PDCoV into PK-15 cells was caveolae-mediated endocytosis that required dynamin and cholesterol; indirect immunofluorescence and shRNA interference further validated these results. (4) Conclusions: In conclusion, PDCoV entry into PK-15 cells depends on caveolae-mediated endocytosis, which requires cholesterol and dynamin. Our finding is the first initial identification of the endocytic pathway of PDCoV in PK-15 cells, providing a theoretical basis for an in-depth understanding of the pathogenic mechanism of PDCoV and the design of new antiviral targets.

Keywords: caveolae-mediated endocytosis; cell entry; clathrin-mediated endocytosis; macropinocytosis; porcine deltacoronavirus (PDCoV)

DOI: https://doi.org/10.3390/v14030496