Palladin Regulates Receptor Clustering and Actin Dynamics in Phagocytosis
Authors: Sun Haimin , Chen Xinlei , Zhu Jiang , Chen Zhu , Chen Saijuan 
Published: October 2019

Abstract
BACKGROUND: Palladin is an actin microfilament associated protein, which together with myotilin and myopalladin form a novel cytoskeletal IgC2 domain protein family. However, little is known about the function of Palladin in myeloid cells. Here, we focus on the function of Palladin in phagocytosis.
METHODS: We used ATRA induced differentiated NB4 cells as neutrophil-like cells, and lenti-viruses were used to build cell lines with palladin or ocrl knockdown and VAMP3-mcherry overexpression. Flow cytometry and immunofluorescence were used to detect the phagocytic ability under conditional opsonins, and the role of palladin knockdown on phagocytic events and F-actin dynamics were observed. CD32 antibodies followed with fluorescent secondary antibody were used as immune complex to stimulate FcγR clustering. We performed the mass spectrometry analysis on the immunoprecipitation (IP) lysate of differentiated NB4 cells, and the protein-protein interactions were confirmed by co-IP experiments; the colocalization and recruitment of different proteins or moleculars were observed under microscopy.
RESULTS: Palladin was up-regulated during ATRA induced differentiation of several AML cell lines, as well as primary mouse bone marrow cells, and its upregulation correlated with increased phagocytic ability. Palladin defective cells showed impaired serum-mediated, IgG- or complement-mediated phagocytosis. The binding ability was measured at 4℃. After 1 hour incubation, ~17% of control cells bound with beads, whereas only ~7% palladin knockdown cells bound with beads, which suggests that Palladin regulated the particle binding. Using serum-opsonized zymosan-FITC as phagocytic targets, we found early phagosome formation, including the pseudopod extension and phagosome closure, was impaired in palladin knockdown cells. However, no significant effect was observed on the recruitment of VAMP3-mcherry, EEA1, Rab7 and LAMP1, so Palladin may not affect the focal exocytosis and phagosome maturation.
The binding defect in Palladin-deficient cells was not attributable to difference in the cell surface expression of Fcγ receptor (FcγR). However, the FcγR clustering was much lower in Palladin-deficient cells. We explored how Palladin influenced the FcγR clustering, and our results showed that Palladin could regulate actin cytoskeleton dynamics and c-Src kinase activation, which resulted in the FcγR clustering.
We also found that in palladin knockdown cells the actin remodeling was detained at both pseudopod extension stage and actin deploymerization stage. The Rac1 were accumulated in higher amount in the blocked cups formed in palladin KD cells, while no significant difference in Cdc42 recruitment. The recruitment intensity of Apr3 was higher in control cells than palladin KD cells. These results suggested that Palladin participated in the actin dynamics during phagocytic cup formation.
We found OCRL is a new Palladin-interacted protein. Palladin interacted with OCRL's 5PPase domain through its third IgC2 domain. Palladin depletion caused a decrease of ~30% OCRL recruitment, retention of PI(4,5)P2, as well as more F-actin at phagosome. These observation suggested that Palladin regulated the recruitment of OCRL at sites of phagosome, and might play an essential role in regulating the hydrolysis of PI(4,5)P2, actin depolymerization and the completion of phagosome closure.
CONCLUSIONS: We identify the role of Palladin in phagocytic receptor clustering and Palladin as an early coordinator in actin dynamics during phagosome formation in myeloid cells.

Disclosures
No relevant conflicts of interest to declare.