Elsevier

Cellular Signalling

Volume 21, Issue 1, January 2009, Pages 1-6
Cellular Signalling

Review
Clathrin-independent endocytosis: A unique platform for cell signaling and PM remodeling

https://doi.org/10.1016/j.cellsig.2008.06.020Get rights and content

Abstract

There is increasing interest in endocytosis that occurs independently of clathrin coats and the fates of membrane proteins internalized by this mechanism. The appearance of clathrin-independent endocytic and membrane recycling pathways seems to vary with different cell types and cargo molecules. In this review we focus on studies that have been performed using HeLa and COS cells as model systems for understanding this membrane trafficking system. These endosomal membranes contain signaling molecules including H-Ras, Rac1, Arf6 and Rab proteins, and a lipid environment rich in cholesterol and PIP2 providing a unique platform for cell signaling. Furthermore, activation of some of these signaling molecules (H-Ras, Rac and Arf6) can switch the constitutive form of clathrin-independent endocytosis into a stimulated one, associated with PM ruffling and macropinocytosis.

Introduction

Endocytosis is a mechanism for cells to remove ligands, nutrients, and plasma membrane (PM) proteins and lipids from the cell surface, bringing them into the cell interior. Once internalized, the membrane and content of the resulting endosome can meet different fates such as movement to late endosomes and lysosomes for degradation, or recycling back out to the PM. Most of the current research on endocytosis has focused on clathrin-dependent endocytosis (CDE).

The hallmark of CDE is the selective sorting of PM proteins by cytosolic adaptor proteins (APs) into clathrin-coated depressions at the surface prior to formation of the clathrin-coated vesicle. Trans-membrane proteins entering through CDE have sequences in their cytoplasmic domains that bind to the APs and enable their rapid removal from the PM [1]. In addition to APs and clathrin, there are numerous accessory proteins that are involved in CDE [2] including dynamin, a GTPase involved in vesicle scission. Immediately after endocytosis, the clathrin/AP coat is released and the vesicle then fuses with the “classical” early endosomal compartment that is defined by the presence of Rab5 and phosphatidylinositol 3-phosphate (PI3P) (see Fig. 1). Once in the early endosome, membrane proteins, lipids and the fluid content of the endosome are sorted and transported either to the trans-Golgi network, to late endosomes and lysosomes for degradation, or into membrane carriers that recycle back to the PM. The CDE pathway is important not only to facilitate the uptake of nutrients, such as iron-loaded transferrin and LDL, into the cell but also for the rapid internalization of most signaling receptors after ligand binding.

There are alterative mechanisms to bring membrane into the cell independently of clathrin. Endocytosis can occur through structures coated with the caveolin protein, and this caveolae-mediated endocytosis, like CDE, is dependent upon dynamin and is responsible for endocytosis of some proteins that partition into cholesterol-rich membrane domains, especially in endothelial cells. There are also two mechanisms of specialized endocytosis that are strictly dependent on cortical actin: phagocytosis, carried out by specialized cells for internalization of large particles such as bacteria, and macropinocytosis, associated with membrane ruffling and involving internalization of large volumes of extracellular fluid and the associated membrane.

Finally, investigators have been studying endocytosis that occurs independently of both clathrin and dynamin. There is some evidence that there might be distinct mechanisms of clathrin-independent endocytosis (CIE) depending upon the cargo and the cell type. The reader is referred to a thorough discussion of this in reviews by Mayor and Pagano [3] and Sandvig et al. [4]. Here we will confine our discussion to the CIE pathway that we and others have been studying in HeLa and COS cells that we believe can serve as a model system for understanding these alternative endosomal systems. By “CIE”, we are referring to the mechanism of endocytosis and when we refer to “CIE pathways” we are referring not only to the mode of endocytosis but also the itinerary and fates of proteins and lipids that are associated with these membranes.

Section snippets

CIE in HeLa cells — unique sets of cargo, regulatory GTPases and membrane lipids

For more than ten years, we have been studying a CIE pathway in HeLa cells that is clearly distinct from, yet intersects with, the CDE pathway (Fig. 1). A variety of endogenous proteins thought to reside only at the PM have been identified that travel into the cell along this pathway including the major histocompatibility Class I protein (MHCI) [5], [6], integrins [7], [8], K channels [9], E-cadherin [10], syndecan 1 [11] and CD59, a protein anchored to the membrane by a glycosylphosphatidyl

Macropinocytosis is a stimulated form of CIE

An intriguing feature of CIE that sets it apart from CDE is that it can operate constitutively in “resting” cells but, upon stimulation, the architecture of the pathway can change to internalization by macropinocytosis during PM ruffling (Fig. 1). In HeLa and COS cells, this switch to macropinocytosis involves the same cargo proteins and membranes involved in CIE. We have found that this transformation can be initiated through the activation of a number of those signaling molecules that travel

Endosomal recycling is required for PM remodeling and related to regulated exocytosis

Regardless of whether membrane enters via the constitutive or stimulated macropinocytic pathway, endosomal membranes are recycled back out to the PM in distinctive carriers and exocytosis is dependent upon the factors listed in Fig. 1. In the case of macropinocytosis, it appears as though the recycling occurs rapidly such that the tubular nature of the recycling endosomes is not observed unless recycling is blocked, for instance by inhibitors of actin polymerization or dominant negative Arf6.

Concluding remarks

In this review we have focused on studies that examined how a CIE membrane system that exists in HeLa and COS cells is regulated and impacts cell surface architecture. There are many questions still to answer in these model systems such as: To what extent are the trafficking pathways altered when the pathway is shifted from constitutive to stimulated macropinocytosis? What is the relationship of the CIE to the CDE pathway? It is important to emphasize that the model depicted in Fig. 1 is meant

Acknowledgements

We thank Ed Korn and Craig Eyster for comments. This work was supported by the Intramural Research Program of the National Heart, Lung and Blood Institute, NIH.

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