Summary-Paper 57

25 Sep 2025
by Richa
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Summary- paper 57: ARF1 compartments direct cargo flow via maturation into recycling endosomes

Alexander Stockhammer, Petia Adarska, Vini Natalia, Anja Heuhsen, Antonia Klemt, Gresy Bregu, Shelly Harel, Carmen Rodilla-Ramirez, Carissa Spalt, Ece Özsoy, Paula Leupold, Alica Grindel, Eleanor Fox, Joy Orezimena Mejedo, Amin Zehtabian, Helge Ewers, Dmytro Puchkov, Volker Haucke & Francesca Bottanelli

Nature Cell Biology, 2024

Questions/gaps addressed:

  • What are the mechanisms and dynamics of proteins sorting out of the Golgi and within the endo-lysosomal system?

Key methods:

  • CRISPR-Cas9 knock-in for endogenous tagging
  • 3D correlative light electron microscopy 3D-CLEM, super-resolution stimulated emission depletion (STED) microscopy.
  • retention using selective hooks (RUSH) system to release a pulse of various secretory cargoes from the ER: RUSH reporters: transferrin receptor (TfR, transmembrane protein), a LAMP1 variant lacking the endocytic motif in its cytoplasmic tail and thus fails to be endocytosed after deposition to the PM (LAMP1Δ, transmembrane protein), TNF (transmembrane protein), VSV-G (transmembrane protein) and a soluble SNAP reporter (sSNAP)

Major takeaways:

  • ARF1 compartments as the major site of clathrin recruitment. ARF1 compartments are almost always associated with non-endocytic clathrin (positive for Arf1, Clathrin, not for AP2). Clathrin localizes at the fission site on ARF1 compartments, suggesting that clathrin and associated machinery may be responsible for the recruitment of fission factors.
  • Both AP1 and AP3 localize to segregated nanodomains on ARF1 compartments. ARF1 compartments in the perinuclear area emerging from the Golgi were only positive for AP-1. Arf1 tubules in the cell periphery were positive for both AP-1 and AP-3. Distinct from fixed cell studies, they found in living gene-edited cells only around 16% of total AP-1 and 6% of total AP-3 puncta, but 80% of the total AP-4 puncta were confined to the perinuclear/TGN area.
  • do both AP-1 and AP-3 can recruit clathrin in living cells?: AP-1 and clathrin on ARF1 compartments were always co-localized, but AP-3 and clathrin localized to segregated nanodomains on ARF1 compartments. AP-1 may be responsible for the recruitment of clathrin and fission machinery to ARF1 compartments. In AP1µA KOs, clathrin recruitment to peripheral ARF1 compartments, but not to the Golgi, was impaired.
  • ARF1 and Rab6 (membrane marker for tubular Golgi-derived carriers mediating direct transport to the PM), do not colocalize at the periphery, but significantly at the Golgi. At the Golgi, about half of the Golgi-derived compartments are positive for ARF1 and Rab6, while the remaining half are Rab6-only carriers devoid of AP-1–functionally distinct classes of Rab6 carriers? Rab6-only tubules may be the direct Golgi-to-PM carriers
  • Also looked at ARF1 and Rab7 (late endosome; LE), ARF1 and SNX1 (sorting endosome, SE), Rab11 (RE) or transiently overexpressed Rab5 (early endosome; EE). ARF1 compartments are not defined by endosomal markers, but partial colocalization was observed with Rab11, RE marker Rab GTPase
  • ARF1 and and Rab11 (RE): partial colocalization, AP-1 and clathrin are seen on ARF1 compartments and on a subpopulation of double-positive Rab11-ARF1 compartments. overlap between ARF1/AP-1/Rab11 revealed a stronger association of AP-1 to ARF1 compartments compared with REs. AP-1 located at the interface of ARF1 compartments and REs in cases where both compartments were found to interact
  • Do ARF1 compartments mature into REs to potentially deliver their content to the PM?: likely, double-positive (ARF1, Rab11) compartments shed their ARF1 coat and acquired more Rab11 over time. ARF1 compartments do not fuse with the PM1 but shed ARF1 to mature into REs.
  • ARF1 and cargos: RUSH system: all reporter cargoes exited the Golgi in ARF1/clathrin-positive compartments that did not harbour AP-3. Cargo first fills ARF1 compartments before transitioning to REs (Rab11). ARF1 compartments containing secretory cargo emerge from the Golgi and over time mature into REs before being able to deliver their content to the PM. In AP1µA KO, retained in long-aberrant perinuclear tubules that were positive for both ARF1 and Rab11. AP-1 may be required for secretory ARF1 compartments to shed ARF1 and mature into REs.
  • AP-1 stays associated with ARF1 compartments during the maturation process and dissociates from the membrane once maturation is complete. ARF1 compartments mediate endocytic recycling via maturation into REs, a process that depends on AP-1.
  • ARF1 compartments are not simply tubulo-vesicular compartments derived from EEs but rather a stand-alone organelle.
  • AP1 and aberrant secretory ARF1 compartment containing anterograde cargo? speculate AP-1 is responsible for the recruitment of yet-to-be identified fission factors.