Distinct mechanisms of clathrin-independent endocytosis have unique sphingolipid requirements

Sphingolipids (SLs) play important roles in membrane structure and cell function. Here, we examine the SL requirements of various endocytic mechanisms using a mutant cell line and pharmacological inhibitors to disrupt SL biosynthesis. First, we demonstrated that in Chinese hamster ovary cells we could distinguish three distinct mechanisms of clathrin-independent endocytosis (caveolar, RhoA, and Cdc42 dependent) which differed in cargo, sensitivity to pharmacological agents, and dominant negative proteins. General depletion of SLs inhibited endocytosis by each clathrin-independent mechanism, whereas clathrin-dependent uptake was unaffected. Depletion of glycosphingolipids (GSLs; a subgroup of SLs) selectively blocked caveolar endocytosis and decreased caveolin-1 and caveolae at the plasma membrane. Caveolar endocytosis and PM caveolae could be restored in GSL-depleted cells by acute addition of exogenous GSLs. Disruption of RhoA- and Cdc42-regulated endocytosis by SL depletion was shown to be related to decreased targeting of these Rho proteins to the plasma membrane and could be partially restored by exogenous sphingomyelin but not GSLs. Both the in vivo membrane targeting and in vitro binding to artificial lipid vesicles of RhoA and Cdc42 were shown to be dependent upon sphingomyelin. These results provide the first evidence that SLs are differentially required for distinct mechanisms of clathrin-independent endocytosis.     

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

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 PIP(2) 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.     

H-Ras signaling and K-Ras signaling are differentially dependent on endocytosis

Endocytosis is required for efficient mitogen-activated protein kinase (MAPK) activation by activated growth factor receptors. We examined if H-Ras and K-Ras proteins, which are distributed across different plasma membrane microdomains, have equal access to the endocytic compartment and whether this access is necessary for downstream signaling. Inhibition of endocytosis by dominant interfering dynamin-K44A blocked H-Ras but not K-Ras-mediated PC12 cell differentiation and selectively inhibited H-Ras- but not K-Ras-mediated Raf-1 activation in BHK cells. H-Ras- but not K-Ras-mediated Raf-1 activation was also selectively dependent on phosphoinositide 3-kinase activity. Stimulation of endocytosis and endocytic recycling by wild-type Rab5 potentiated H-Ras-mediated Raf-1 activation. In contrast, Rab5-Q79L, which stimulates endocytosis but not endocytic recycling, redistributed activated H-Ras from the plasma membrane into enlarged endosomes and inhibited H-Ras-mediated Raf-1 activation. Rab5-Q79L expression did not cause the accumulation of wild-type H-Ras in enlarged endosomes. Expression of wild-type Rab5 or Rab5-Q79L increased the specific activity of K-Ras-activated Raf-1 but did not result in any redistribution of K-Ras from the plasma membrane to endosomes. These results show that H-Ras but not K-Ras signaling though the Raf/MEK/MAPK cascade requires endocytosis and endocytic recycling. The data also suggest a mechanism for returning Raf-1 to the cytosol after plasma membrane recruitment.     

Pathways of clathrin-independent endocytosis

There are numerous ways that endocytic cargo molecules may be internalized from the surface of eukaryotic cells. In addition to the classical clathrin-dependent mechanism of endocytosis, several pathways that do not use a clathrin coat are emerging. These pathways transport a diverse array of cargoes and are sometimes hijacked by bacteria and viruses to gain access to the host cell. Here, we review our current understanding of various clathrin-independent mechanisms of endocytosis and propose a classification scheme to help organize the data in this complex and evolving field.     

Driving membrane curvature in clathrin-dependent and clathrin-independent endocytosis

Cellular activity depends to a large extent on membrane bilayer dynamics. Many processes, such as organelle biogenesis and vesicular transport, rely on alterations in membrane structure and shape. It is now widely accepted that intracellular membrane curvature generation and remodelling is mediated and regulated by protein action, and the mechanisms behind the processes are currently being revealed. Here, we will briefly discuss the key principles of membrane deformation and focus on different endocytic events that use various kinds of proteins to shape the plasma membrane into transport carriers. The entry routes are adopted to make sure that a vast variety of molecules on the cell surface can be regulated by endocytosis. The principles for membrane sculpting of endocytic carriers can be viewed either from a perspective of rigid coat budding or of flexible opportunistic budding. We will discuss these principles and their implications, focusing on clathrin-dependent and -independent carrier formation and the proteins involved in the respective pathways.     

Membrane protrusion powers clathrin-independent endocytosis of interleukin-2 receptor

Endocytosis controls many functions including nutrient uptake, cell division, migration and signal transduction. A clathrin- and caveolin-independent endocytosis pathway is used by important physiological cargos, including interleukin-2 receptors (IL-2R). However, this process lacks morphological and dynamic data. Our electron microscopy (EM) and tomography studies reveal that IL-2R-pits and vesicles are initiated at the base of protrusions. We identify the WAVE complex as a specific endocytic actor. The WAVE complex interacts with IL-2R, via a WAVE-interacting receptor sequence (WIRS) present in the receptor polypeptide, and allows for receptor clustering close to membrane protrusions. In addition, using total internal reflection fluorescent microscopy (TIRF) and automated analysis we demonstrate that two timely distinct bursts of actin polymerization are required during IL-2R uptake, promoted first by the WAVE complex and then by N-WASP. Finally, our data reveal that dynamin acts as a transition controller for the recruitment of Arp2/3 activators required for IL-2R endocytosis. Altogether, our work identifies the spatio-temporal specific role of factors initiating clathrin-independent endocytosis by a unique mechanism that does not depend on the deformation of a flat membrane, but rather on that of membrane protrusions.     

The Ras-PI3K signaling pathway is involved in clathrin-independent endocytosis and the internalization of influenza viruses

Background

Influenza virus infection causes highly contagious, severe respiratory disorders and gives rise to thousands of deaths every year; however, the efficacy of currently approved defense strategies, including vaccines and neuraminidase inhibitors, is limited because the virus frequently acquires resistance via antigen drift and reassortment. It is therefore important to establish a novel, effective therapeutic strategy that is effective irrespective of viral subtype.

Methodology/Principal Findings

Here, we identify the Ras–phosphoinositide 3-kinase (PI3K) signaling pathway as a host-cell regulatory mechanism for influenza virus entry. The binding of Ras to PI3K is specifically involved in clathrin-independent endocytosis, endosomal maturation, and intracellular transport of viruses, which result in decreased infectious efficacy of different subtypes of influenza viruses in cells lacking the Ras–PI3K interaction. Moreover, influenza virus infection indeed triggered Ras activation and subsequent PI3K activation in early endosomes.

Conclusions/Significance

Taken together, these results demonstrate that the Ras–PI3K signaling axis acts as a host-oriented mechanism for viral internalization. Given that virus incorporation is a process conserved among virus subtypes and species, this signaling pathway may provide a target for potent, well-tolerated prophylactics and therapeutics against a broad range of viruses.     

A distinct class of endosome mediates clathrin-independent endocytosis to the Golgi complex

Mammalian cells endocytose a variety of proteins and lipids without utilising clathrin-coated pits. Detailed molecular mechanisms for clathrin-independent endocytosis are unclear. Several markers for this process, including glycosphingolipid-binding bacterial toxin subunits such as cholera toxin B subunit (CTxB), and glycosyl-phosphatidyl-inositol (GPI)-anchored proteins, are found in detergent-resistant membrane fractions (DRMs), or 'lipid rafts'. The Golgi complex constitutes one principal intracellular destination for these markers. Uptake of both CTxB and GPI-anchored proteins may involve caveolae, small invaginations in the plasma membrane (PM). However, the identity of intermediate organelles involved in PM to Golgi trafficking, as well as the function of caveolins, defining protein components of caveolae, are unclear. This paper shows that molecules which partition into DRMs and are endocytosed in a clathrin-independent fashion, accumulate in a discrete population of endosomes en route to the Golgi complex. These endosomes are devoid of markers for classical early and recycling endosomes, but do contain caveolin-1. Caveolin-1-positive endosomes are sites for the sorting of caveolin-1 away from Golgi-bound cargoes, although caveolin-1 itself is unlikely to have a direct function in PM to Golgi transport.     

Cortactin and dynamin are required for the clathrin-independent endocytosis of gammac cytokine receptor

Endocytosis is critical for many cellular functions. We show that endocytosis of the common gammac cytokine receptor is clathrin independent by using a dominant-negative mutant of Eps15 or RNA interference to knock down clathrin heavy chain. This pathway is synaptojanin independent and requires the GTPase dynamin. In addition, this process requires actin polymerization. To further characterize the function of dynamin in clathrin-independent endocytosis, in particular its connection with the actin cytoskeleton, we focused on dynamin-binding proteins that interact with F-actin. We compared the involvement of these proteins in the clathrin-dependent and -independent pathways. Thus, we observed that intersectin, syndapin, and mAbp1, which are necessary for the uptake of transferrin (Tf), a marker of the clathrin route, are not required for gammac receptor endocytosis. Strikingly, cortactin is needed for both gammac and Tf internalizations. These results reveal the ubiquitous action of cortactin in internalization processes and suggest its role as a linker between actin dynamics and clathrin-dependent and -independent endocytosis.     

A critical role for endocytosis in Wnt signaling

Background  

The Wnt signaling pathway regulates many processes during embryonic development, including axis specification, organogenesis, angiogenesis, and stem cell proliferation. Wnt signaling has also been implicated in a number of cancers, bone density maintenance, and neurological conditions during adulthood. While numerous Wnts, their cognate receptors of the Frizzled and Arrow/LRP5/6 families and downstream pathway components have been identified, little is known about the initial events occurring directly after receptor activation.     

The CD20 homologue MS4A4 directs trafficking of KIT toward clathrin-independent endocytosis pathways and thus regulates receptor signaling and recycling

MS4A family members differentially regulate the cell cycle, and aberrant, or loss of, expression of MS4A family proteins has been observed in colon and lung cancer. However, the precise functions of MS4A family proteins and their mechanistic interactions remain unsolved. Here we report that MS4A4 facilitates trafficking of the receptor tyrosine kinase KIT through endocytic recycling rather than degradation pathways by a mechanism that involves recruitment of KIT to caveolin-1–enriched microdomains. Silencing of MS4A4 in human mast cells altered ligand-induced KIT endocytosis pathways and reduced receptor recycling to the cell surface, thus promoting KIT signaling in the endosomes while reducing that in the plasma membrane, as exemplified by Akt and PLCγ1 phosphorylation, respectively. The altered endocytic trafficking of KIT also resulted in an increase in SCF-induced mast cell proliferation and migration, which may reflect altered signaling in these cells. Our data reveal a novel function for MS4A family proteins in regulating trafficking and signaling, which could have implications in both proliferative and immunological diseases.     

Molecules internalized by clathrin-independent endocytosis are delivered to endosomes containing transferrin receptors

We have previously demonstrated that the preendosomal compartment in addition to clathrin-coated vesicles, comprises distinct nonclathrin coated endocytic vesicles mediating clathrin-independent endocytosis (Hansen, S. H., K. Sandvig, and B. van Deurs. 1991. J. Cell Biol. 113:731-741). Using K+ depletion in HEp-2 cells to block clathrin- dependent but not clathrin-independent endocytosis, we have now traced the intracellular routing of these nonclathrin coated vesicles to see whether molecules internalized by clathrin-independent endocytosis are delivered to a unique compartment or whether they reach the same early and late endosomes as encountered by molecules internalized with high efficiency through clathrin-coated pits and vesicles. We find that Con A-gold internalized by clathrin-independent endocytosis is delivered to endosomes containing transferrin receptors. After incubation of K(+)- depleted cells with Con A-gold for 15 min, approximately 75% of Con A- gold in endosomes is colocalized with transferrin receptors. Endosomes containing only Con A-gold may be accounted for either by depletion of existing endosomes for transferrin receptors or by de novo generation of endosomes. Cationized gold and BSA-gold internalized in K(+)- depleted cells are also delivered to endosomes containing transferrin receptors. h-lamp-1-enriched compartments are only reached occasionally within 30 min in K(+)-depleted as well as in control cells. Thus, preendosomal vesicles generated by clathrin-independent endocytosis do not fuse to any marked degree with late endocytic compartments. These data show that in HEp-2 cells, molecules endocytosed without clathrin are delivered to the same endosomes as reached by transferrin receptors internalized through clathrin-coated pits.     

Differentiation between clathrin-dependent and clathrin-independent endocytosis by means of membrane fluidity measurements

The fluorescence probe [1-(4-trimethylammonium]-6-phenyl-1,3,5-hexatriene (TMA-DPH) displays properties relevant for both monitoring endocytosis kinetics and assessing membrane fluidity by fluorescence-anisotropy measurements (1). Thus, it is, possible with this probe to follow the evolution of membrane fluidity during endocytosis, from the very beginning of the process, i.e., the formation of endocytic vesicles. In most cases, endocytosis is known to start with clathrin-coated vesicles. Still, there are more and more arguments in favor of a complementary endocytic pathway without clathrin. In this article we present membrane-fluidity data for very early endocytosis, which allow an upper limit to be determined for the contribution of a putative nonclathrin pathway. We show that this limit is markedly higher for bone marrow-derived macrophages than for mouse fibroblasts of the L929 cell line.     

Transfer of M2 muscarinic acetylcholine receptors to clathrin-derived early endosomes following clathrin-independent endocytosis

Upon agonist stimulation, many G protein-coupled receptors such as beta(2)-adrenergic receptors are internalized via beta-arrestin- and clathrin-dependent mechanisms, whereas others, like M(2) muscarinic acetylcholine receptors (mAChRs), are internalized by clathrin- and arrestin-independent mechanisms. To gain further insight into the mechanisms that regulate M(2) mAChR endocytosis, we investigated the post-endocytic trafficking of M(2) mAChRs in HeLa cells and the role of the ADP-ribosylation factor 6 (Arf6) GTPase in regulating M(2) mAChR internalization. Here, we report that M(2) mAChRs are rapidly internalized by a clathrin-independent pathway that is inhibited up to 50% by expression of either GTPase-defective Arf6 Q67L or an upstream Arf6 activator, Galpha(q) Q209L. In contrast, M(2) mAChR internalization was not affected by expression of dominant-negative dynamin 2 K44A, which is a known inhibitor of clathrin-dependent endocytosis. Nevertheless, M(2) mAChRs, which are initially internalized in structures that lack clathrin-dependent endosomal markers, quickly localize to endosomes that contain the clathrin-dependent, early endosomal markers early endosome autoantigen-1, transferrin receptor, and GTPase-defective Rab5 Q79L, which is known to swell early endosomal compartments. These results suggest that M(2) mAChRs initially internalize via an Arf6-associated, clathrin-independent pathway but then quickly merge with the clathrin endocytic pathway at the level of early endosomes.     

Uptake of fluorescent nano beads into BY2-cells involves clathrin-dependent and clathrin-independent endocytosis

To follow endocytosis in BY-2 cells we made use of fluorescent nano beads. Beads with 20 nm in diameter were internalised rapidly and accumulated partially in compartments also labelled by the endocytic marker FM4-64. Studies in BY-2 cells and protoplasts revealed that larger beads (100 nm) were excluded from uptake into turgescent and plasmolysed cells while protoplasts were able to internalise beads with a diameter of up to 1000 nm. Endocytosis of beads was only partially inhibited by the clathrin-specific inhibitor Ikarugamycin and strongly blocked by wortmannin. These results imply that uptake of beads involves clathrin-dependent and clathrin-independent endocytic mechanisms and supports the hypothesis that clathrin-independent endocytosis plays a general role in plants.     

Ca2+ triggers a novel clathrin-independent but actin-dependent fast endocytosis in pancreatic beta cells

The existence of clathrin-independent recycling of secretory vesicles has been controversial. By combining patch-clamp capacitance recording, optical methods and specific molecular interventions, we dissect two types of mechanistically different endocytosis in pancreatic β cells, both of which require GTP and dynamin. The fast one is a novel clathrin-independent but actin-dependent endocytosis that is triggered by high cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). Large fluorescent dextran (10 nm in diameter) was able to be internalized by this pathway, indicating that it was not likely to be 'kiss and run'. The slow endocytosis is a clathrin-dependent process in which actin plays a complementary role. For the first time, we show that the rate constants for both types of endocytosis exhibit supralinear dependence on increase in [Ca²⁺]_i. Compared with the slow endocytosis, higher [Ca²⁺]_i level was required to fully accelerate the fast one, indicative of distinct Ca²⁺ sensors for different endocytosis. In the end, we show that physiologically relevant stimulation induces clathrin-independent endocytosis in intact β cells, implying that it may contribute to the normal recycling of secretory vesicles in vivo .     

Reconstitution of clathrin-independent endocytosis at the apical domain of permeabilized MDCK II cells: requirement for a Rho-family GTPase

This paper studies the endocytosis of ricin at the apical pole of polarized MDCK II cells after permeabilization of the cells basolaterally with streptolysin O. Ricin endocytosis after the addition of cytosol with an ATP-regenerating system was 2-3-fold higher than after the addition of a transport medium. A similar increase in ricin endocytosis was obtained by reconstitution of dialyzed cytosol with the nonhydrolyzable GTP analog, GTP gamma S, in the presence of an ATP-regenerating system. The nonhydrolyzable GDP analog, GDP beta S, did not increase ricin uptake. In contrast to the data obtained with ricin, GTP gamma S was found to inhibit apical transferrin uptake in MDCK II cells transfected with the human transferrin receptor, and the data thus imply that GTP gamma S supports clathrin-independent endocytosis. Electron microscopy (EM) demonstrated that free endocytic vesicles were formed from the apical pole of permeabilized MDCK II cells in the presence of GTP gamma S and that both a ricin-HRP conjugate, HRP, and cationized gold were endocytosed. Ricin endocytosis in the presence of intact cytosol, as well as GTP gamma S-stimulated ricin uptake, was inhibited by Clostridium botulinum C3 transferase, an enzyme found to inactivate Rho proteins. The data demonstrate that apical clathrin-independent endocytosis functions in the presence of GTP gamma S, and suggest that one or more of the small GTP binding proteins of the Rho family is involved in regulation of the apical clathrin-independent endocytosis in MDCK II cells.