Summary-Paper 14

10 Feb 2023
by Richa
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Summary- paper 14: Calcium levels in the Golgi complex regulate clustering and apical sorting of GPI-APs in polarized epithelial cells

Stéphanie Lebreton, Simona Paladino, Dandan Liu, Maria Nitti, Julia von Blume, Paolo Pinton, Chiara Zurzolo

PNAS, 2021

Questions/gaps addressed:

  • Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are lipid-associated luminal secretory cargos. Distinct clustering mechanisms drive their sorting out of the Golgi apparatus to the cell surface in polarized cells and non-polarized cells. What are the mechanisms of GPI-AP apical sorting in the Golgi apparatus?

  • Golgi exit of various cargos is altered in cells treated with drugs either depolymerizing/ stabilizing actin filaments/ mutants affecting actin function. High calcium levels in the Golgi are important for sorting of some secreted soluble proteins. Possible role of the actin cytoskeleton and of calcium levels in the Golgi?

Major hypotheses:

  • The amount of calcium in the Golgi lumen regulates the formation of GPI-AP homoclusters in polarized epithelial cells.

Key methods:

  • Polarized MDCK (Madin-Darby Canine Kidney) cells treated with ionomycin to deplete calcium ions from the Golgi (preferentially depletes Golgi luminal calcium) amd monitored effect on localization of a model apical GPI-AP, GFP-FR. Observed the treatment resulted in reduced accumulation of HMW or multimeric complexes of GFP-FR.

  • Measured the calcium concentration in the Golgi by using a calcium-sensitive photoprotein (Golgi-aequorin chimera). Found calcium levels in the Golgi apparatus of polarized MDCK cells are slightly higher compared to the Golgi of nonpolarized MDCK cells.

  • Effect of two groups of phosphorylation-type calcium pumps, SERCA and SPCA1. SPCA1 protein levels increase with the establishment of polarity. shRNA KD of SPCA1 leads to reduction in the concentration of calcium in the Golgi using the calcium sensor, and reduced the homoclustering of GFP-FR.

  • Transport kinetics of GFP-FR using time-lapse confocal microscopy upon SPCA1 KD to look at Golgi export using temperature block assays. KD affected both the Golgi exit and the apical sorting. Cab45, a Golgi (TGN) luminal protein, oligomerizes upon calcium binding and required for protein sorting. KD of Cab45 lead to missorting of GFP-FR to the basolateral membrane. Observed similar effects on endogenous GPI-AP, PLAP (placental alkaline phosphatase).

  • Fluorescent-conjugated bacterial toxin aerolysin, which binds with high affinity GPI-APs to monitor surface distribution of endogenous GPI-APs upon Cab45 KD, observed missorting to basolateral surface.

  • Treatment with latrunculin A to perturb actin cytoskeleton and monitored GFP-FR localization and clustering, no effect of actin perturbation on the oligomeric status.

Major takeaways:

  • While high cholesterol concentration is necessary for the clustering of GPI-APs, this is not sufficient for their apical sorting.

  • Golgi organization of GPI-APs is drastically perturbed upon calcium depletion and that the amount of calcium in the Golgi cisternae is critical for the formation of GPI-AP homoclusters.

  • Down-regulation of the TGN calcium/manganese pump, SPCA1 (secretory pathway Ca(2+)-ATPase pump type 1) or Cab45, a calcium-binding luminal Golgi resident protein, impairs the oligomerization of GPI-APs in the Golgi complex and leads to their missorting to the basolateral surface but does not affect apical or basolateral transmembrane proteins, or basolateral GPI-APs such as PrP.

  • Levels of SPCA1 increase during polarization, while levels of Cab45 decrease (but oligomerization increases) during polarization.

  • Calcium-binding proteins, such as calmodulin, are thought to mediate fusion between yeast vacuoles, the later steps of fusion vesicle trafficking and endosome fusion. Do Golgi luminal calcium-binding proteins, Cab45 and p54/NEFA (yeast SSP120), work in similar ways?

  • The calcium-dependent mechanism for GPI-AP sorting is dominant over dependence on Golgi cholesterol levels, apical sorting of GPI-APs. What is the interplay between Golgi cholesterol levels and Calcium levels?