SPIN90 Knockdown Attenuates the Formation and Movement of Endosomal Vesicles in the Early Stages of Epidermal Growth Factor Receptor Endocytosis

Abstract

The finding that SPIN90 colocalizes with epidermal growth factor (EGF) in EEA1-positive endosomes prompted us to investigate the role of SPIN90 in endocytosis of the EGF receptor (EGFR). In the present study, we demonstrated that SPIN90 participates in the early stages of endocytosis, including vesicle formation and trafficking. Stable HeLa cells with knockdown of SPIN90 displayed significantly higher levels of surface EGFR than control cells. Analysis of the abundance and cellular distribution of EGFR via electron microscopy revealed that SPIN90 knockdown cells contain residual EGFR at cell membranes and fewer EGFR-containing endosomes, both features that reflect reduced endosome formation. The delayed early endosomal targeting capacity of SPIN90 knockdown cells led to increased EGFR stability, consistent with the observed accumulation of EGFR at the membrane. Small endosome sizes and reduced endosome formation in SPIN90 knockdown cells, observed using fluorescent confocal microscopy, strongly supported the involvement of SPIN90 in endocytosis of EGFR. Overexpression of SPIN90 variants, particularly the SH3, PRD, and CC (positions 643 - 722) domains, resulted in aberrant morphology of Rab5-positive endosomes (detected as small spots located near the cell membrane) and defects in endosomal movement. These findings clearly suggest that SPIN90 participates in the formation and movement of endosomes. Consistent with this, SPIN90 knockdown enhanced cell proliferation. The delay in EGFR endocytosis effectively increased the levels of endosomal EGFR, which triggered activation of ERK1/2 and cell proliferation via upregulation of cyclin D1. Collectively, our findings suggest that SPIN90 contributes to the formation and movement of endosomal vesicles, and modulates the stability of EGFR protein, which affects cell cycle progression via regulation of the activities of downstream proteins, such as ERK1/2, after EGF stimulation.

Correlative single‐molecule localization microscopy and electron tomography reveals endosome nanoscale domains

“…Tfn was primarily located in recycling tubules with often complex, interwoven geometries, which are not resolvable by SMLM alone. These observations are in accordance with previously reported findings based on EM analyses . However, neither EGF nor Tfn were exclusively confined to one particular compartment since we also found disperse Tfn signal on the limiting membrane of endosomes, and EGF signal neither associated with ILVs nor distinct microdomains.…”

Dip1 Co-opts Features of Branching Nucleation to Create Linear Actin Filaments that Activate WASP-Bound Arp2/3 Complex

“…This phenotype is identical to the dip1 Δ phenotype in fission yeast [5], and is consistent with a model in which Ldb17 initiates new patches by providing preformed filaments to activate WASP-bound Arp2/3 complex. Less is known about the in vivo function of the mammalian WDS protein, SPIN90, but experiments suggest that it interacts with endocytic proteins, and contributes to uptake of at least one endocytic cargo, EGFR [29]. In addition to its roles in endocytosis, some studies suggest SPIN90 may play a role in assembly of actin in lamellipodia.…”

Genome-Wide Analysis of Protein-Coding Variants in Leprosy

“…It is a part of FCG receptor-dependent phagocytosis pathway and is implicated in many functional processes, such as assembly and maintenance of sarcomeres and stress fiber formation (Lim et al, 2001;Satoh and Tominaga, 2001). The rare variant rs145562243 (p.R176Q) was located in the proline-rich region (PRD) of the NCKIPSD N-terminus, whose overexpression was found to be related to abnormalities in vesicle formation and trafficking, leading to the defective endocytosis of FCG receptor (Oh et al, 2013). Together with the identification of RAB32 (Zhang et al, 2011), which has been shown to be involved in autophagy and phagocytotic digestion of bacteria (Hirota and Tanaka, 2009;Seto et al, 2011;Spano and Galan, 2012), as well as LRRK2 (Zhang et al, 2009) as an interacting partner of RAB32 (Waschbusch et al, 2014) and related to cell death due to autophagic dysfunction and mitochondrial damage (Alegre-Abarrategui et al, 2009;Plowey et al, 2008), the discovery of NCKIPSD highlights the potential involvement of endocytosis/phagocytosis/autophagy in host defense against M. leprae infection.…”