Annexin A8 Regulates Late Endosome Organization and Function

Abstract

Different classes of endosomes exhibit a characteristic intracellular steady-state distribution governed by interactions with the cytoskeleton. Late endosomes, organelles of the degradative lysosomal route, seem to require associated actin filaments for proper localization and function. We show here that the F-actin and phospholipid binding protein annexin A8 is associated specifically with late endosomes. Altering intracellular annexin A8 levels drastically affected the morphology and intracellular distribution of late endosomes. Trafficking through the degradative pathway was delayed in the absence of annexin A8, resulting in attenuated ligand-induced degradation of the epidermal growth factor receptor and prolonged epidermal growth factor-induced activation of mitogen-activated protein kinase. Depletion of annexin A8 reduced the association of late endosomal membranes with actin filaments. These results indicate that the defective cargo transport through the late endocytic pathway and the imbalanced signaling of activated receptors observed in the absence of annexin A8 results from the disturbed association of late endosomal membranes with the actin network, resulting in impaired actin-based late endosome motility.

Annexin A8 controls leukocyte recruitment to activated endothelial cells via cell surface delivery of CD63

“…3C). In accordance with our previous data obtained in HeLa cells 15 , the anxA8-GFP-positive late endosomes were sometimes enlarged.…”

“…Because anxA8 affects late endosome morphology and function in HeLa cells 15 and CD63 and P-selectin are thought to cycle through endosomes and then WPB to reach the endothelial cell surface following activation of WPB exocytosis 18 , we studied the effect of anxA8 on inflammatory P-selectin/CD63 functions. We ablated the protein in HUVEC using anxA8-specific small interfering RNA (siRNA) and non-targeting siRNA as control and examined the effect on primary human polymorphonuclear neutrophil (PMN) recruitment at physiological flow rates.…”

“…To trigger regulated secretion, cells were treated with 100 mM histamine (Sigma-Aldrich) for 20 min in basal medium (M-199; PAA, Pasching, Austria) containing 0.5% BSA, 100 U ml À 1 penicillin and 100 mg ml À 1 streptomycin. siRNA-mediated depletion of anxA8 was achieved by transfecting HUVEC with 200-400 pmol siRNA (5 0 -GGAGCGAGAUUGACUUAAAdTdT-3 0 ) 15 . Control experiments used nontargeting siRNAs (AllStars negative control siRNA; Qiagen, Valencia, CA, USA).…”

“…Therefore, we set out to identify cellular factors required for the delivery of CD63 to WPB, focussing on endosome-associated components. Based on our previous finding showing that annexin A8 (anxA8) is required for late endosomal functions in nonpolarized cells 15 , we hypothesized that this protein might participate in the delivery of CD63 from late endosomes to WPB. AnxA8 is a member of the annexin family of membranebinding proteins that have been implicated in various membranetrafficking steps.…”

“…AnxA8 is a member of the annexin family of membranebinding proteins that have been implicated in various membranetrafficking steps. These include roles for anxA1 and anxA2 in the formation of late multivesicular endosomes (for reviews, see Futter and White 16 , and Gerke et al 17 ) and for anxA8 in maintaining proper late endosome morphology and distribution, most likely by regulating the amount of endosome-associated actin 15 .…”