Cdc42 Regulates Microtubule‐Dependent Golgi Positioning

The molecular mechanisms underlying cytoskeleton‐dependent Golgi positioning are poorly understood. In mammalian cells, the Golgi apparatus is localized near the juxtanuclear centrosome via dynein‐mediated motility along microtubules. Previous studies implicate Cdc42 in regulating dynein‐dependent motility. Here we show that reduced expression of the Cdc42‐specific GTPase‐activating protein, ARHGAP21, inhibits the ability of dispersed Golgi membranes to reposition at the centrosome following nocodazole treatment and washout. Cdc42 regulation of Golgi positioning appears to involve ARF1 and a binding interaction with the vesicle‐coat protein coatomer. We tested whether Cdc42 directly affects motility, as opposed to the formation of a trafficking intermediate, using a Golgi capture and motility assay in permeabilized cells. Disrupting Cdc42 activation or the coatomer/Cdc42 binding interaction stimulated Golgi motility. The coatomer/Cdc42‐sensitive motility was blocked by the addition of an inhibitory dynein antibody. Together, our results reveal that dynein and microtubule‐dependent Golgi positioning is regulated by ARF1‐, coatomer‐, and ARHGAP21‐dependent Cdc42 signaling.     

Facing Inward from Compartment Shores: How Many Pathways were we Looking For?

Protein toxins of the Shiga family have become potent tools in studying a number of intracellular transport events such as endocytosis, the communication between endosomes and the biosynthetic/secretory pathway, and retrograde transport from the Golgi apparatus to the endoplasmic reticulum. It seems clear today that most of these transport events can be explained from the toxins' interactions with cellular factors. This review will primarily focus on the discussion of recent data obtained on Shiga toxin and related toxins. We will point out to what extent the study of these proteins has opened new avenues for the development of intracellular targeting tools.     

Regulation of protein transport from the Golgi complex to the endoplasmic reticulum by CDC42 and N-WASP

Actin is involved in the organization of the Golgi complex and Golgi-to-ER protein transport in mammalian cells. Little, however, is known about the regulation of the Golgi-associated actin cytoskeleton. We provide evidence that Cdc42, a small GTPase that regulates actin dynamics, controls Golgi-to-ER protein transport. We located GFP-Cdc42 in the lateral portions of Golgi cisternae and in COPI-coated and non-coated Golgi-associated transport intermediates. Overexpression of Cdc42 and its activated form Cdc42V12 inhibited the retrograde transport of Shiga toxin from the Golgi complex to the ER, the redistribution of the KDEL receptor, and the ER accumulation of Golgi-resident proteins induced by the active GTP-bound mutant of Sar1 (Sar1[H79G]). Coexpression of wild-type or activated Cdc42 and N-WASP also inhibited Golgi-to-ER transport, but this was not the case in cells expressing Cdc42V12 and N-WASP(Delta WA), a mutant form of N-WASP that lacks Arp2/3 binding. Furthermore, Cdc42V12 recruited GFP-N-WASP to the Golgi complex. We therefore conclude that Cdc42 regulates Golgi-to-ER protein transport in an N-WASP-dependent manner.     

Shiga toxin facilitates its retrograde transport by modifying microtubule dynamics

The bacterial exotoxin Shiga toxin is endocytosed by mammalian host cells and transported retrogradely through the secretory pathway before entering the cytosol. Shiga toxin also increases the levels of microfilaments and microtubules (MTs) upon binding to the cell surface. The purpose for this alteration in cytoskeletal dynamics is unknown. We have investigated whether Shiga toxin-induced changes in MT levels facilitate its intracellular transport. We have tested the effects of the Shiga toxin B subunit (STB) on MT-dependent and -independent transport steps. STB increases the rate of MT-dependent Golgi stack repositioning after nocodazole treatment. It also enhances the MT-dependent accumulation of transferrin in a perinuclear recycling compartment. By contrast, the rate of MT-independent transferrin recycling is not significantly different when STB is present. We found that STB normally requires MTs and dynein for its retrograde transport to the juxtanuclear Golgi complex and that STB increases MT assembly. Furthermore, we find that MT polymerization is limiting for STB transport in cells. These results show that STB-induced changes in cytoskeletal dynamics influence intracellular transport. We conclude that the increased rate of MT assembly upon Shiga toxin binding facilitates the retrograde transport of the toxin through the secretory pathway.     

Pathways followed by ricin and Shiga toxin into cells

The plant toxin ricin and the bacterial toxin Shiga toxin belong to a group of protein toxins that inhibit protein synthesis in cells enzymatically after entry into the cytosol. Ricin and Shiga toxin, which both have an enzymatically active moiety that inactivates ribosomes and a moiety that binds to cell surface receptors, enter the cytosol after binding to the cell surface, endocytosis by different mechanisms, and retrograde transport to the Golgi apparatus and the endoplasmic reticulum (ER). The toxins can be used to investigate the various transport steps involved, both the endocytic mechanisms as well as pathways for retrograde transport to the ER. Recent studies show that not only do several endocytic mechanisms exist in the same cell, but they are not equally sensitive to removal of cholesterol. New data have revealed that there is also more than one pathway leading from endosomes to the Golgi apparatus and retrogradely from the Golgi to the ER. Trafficking of protein toxins along these pathways will be discussed in the present article.     

Geldanamycin Enhances Retrograde Transport of Shiga Toxin in HEp-2 Cells

The heat shock protein 90 (Hsp90) inhibitor geldanamycin (GA) has been shown to alter endosomal sorting, diverting cargo destined for the recycling pathway into the lysosomal pathway. Here we investigated whether GA also affects the sorting of cargo into the retrograde pathway from endosomes to the Golgi apparatus. As a model cargo we used the bacterial toxin Shiga toxin, which exploits the retrograde pathway as an entry route to the cytosol. Indeed, GA treatment of HEp-2 cells strongly increased the Shiga toxin transport to the Golgi apparatus. The enhanced Golgi transport was not due to increased endocytic uptake of the toxin or perturbed recycling, suggesting that GA selectively enhances endosomal sorting into the retrograde pathway. Moreover, GA activated p38 and both inhibitors of p38 or its substrate MK2 partially counteracted the GA-induced increase in Shiga toxin transport. Thus, our data suggest that GA-induced p38 and MK2 activation participate in the increased Shiga toxin transport to the Golgi apparatus.     

CLN3 Deficient Cells Display Defects in the ARF1-Cdc42 Pathway and Actin-Dependent Events

Juvenile Batten disease (juvenile neuronal ceroid lipofuscinosis, JNCL) is a devastating neurodegenerative disease caused by mutations in CLN3, a protein of undefined function. Cell lines derived from patients or mice with CLN3 deficiency have impairments in actin-regulated processes such as endocytosis, autophagy, vesicular trafficking, and cell migration. Here we demonstrate the small GTPase Cdc42 is misregulated in the absence of CLN3, and thus may be a common link to multiple cellular defects. We discover that active Cdc42 (Cdc42-GTP) is elevated in endothelial cells from CLN3 deficient mouse brain, and correlates with enhanced PAK-1 phosphorylation, LIMK membrane recruitment, and altered actin-driven events. We also demonstrate dramatically reduced plasma membrane recruitment of the Cdc42 GTPase activating protein, ARHGAP21. In line with this, GTP-loaded ARF1, an effector of ARHGAP21 recruitment, is depressed. Together these data implicate misregulated ARF1-Cdc42 signaling as a central defect in JNCL cells, which in-turn impairs various cell functions. Furthermore our findings support concerted action of ARF1, ARHGAP21, and Cdc42 to regulate fluid phase endocytosis in mammalian cells. The ARF1-Cdc42 pathway presents a promising new avenue for JNCL therapeutic development.     

Coatomer-bound Cdc42 regulates dynein recruitment to COPI vesicles

Cytoskeletal dynamics at the Golgi apparatus are regulated in part through a binding interaction between the Golgi-vesicle coat protein, coatomer, and the regulatory GTP-binding protein Cdc42 (Wu, W.J., J.W. Erickson, R. Lin, and R.A. Cerione. 2000. Nature. 405:800-804; Fucini, R.V., J.L. Chen, C. Sharma, M.M. Kessels, and M. Stamnes. 2002. Mol. Biol. Cell. 13:621-631). The precise role of this complex has not been determined. We have analyzed the protein composition of Golgi-derived coat protomer I (COPI)-coated vesicles after activating or inhibiting signaling through coatomer-bound Cdc42. We show that Cdc42 has profound effects on the recruitment of dynein to COPI vesicles. Cdc42, when bound to coatomer, inhibits dynein binding to COPI vesicles whereas preventing the coatomer-Cdc42 interaction stimulates dynein binding. Dynein recruitment was found to involve actin dynamics and dynactin. Reclustering of nocodazole-dispersed Golgi stacks and microtubule/dynein-dependent ER-to-Golgi transport are both sensitive to disrupting Cdc42 mediated signaling. By contrast, dynein-independent transport to the Golgi complex is insensitive to mutant Cdc42. We propose a model for how proper temporal regulation of motor-based vesicle translocation could be coupled to the completion of vesicle formation.     

Retrograde transport from the Golgi complex to the ER of both Shiga toxin and the nontoxic Shiga B-fragment is regulated by butyric acid and cAMP

Endocytosed Shiga toxin is transported from the Golgi complex to the endoplasmic reticulum in butyric acid-treated A431 cells. We here examine the extent of this retrograde transport and its regulation. The short B fragment of Shiga toxin is sufficient for transport to the ER. The B fragment of cholera toxin, which also binds to glycolipids, is transported to all the Golgi cisterns, but cannot be localized in the ER even after butyric acid treatment. Under all conditions the toxic protein ricin was found predominantly in the trans-Golgi network. There is no transport of endocytosed fluid to the Golgi apparatus or to the ER even after butyric acid treatment and in the presence of Shiga toxin, indicating that transport to the ER, through the trans-Golgi network and the cisterns of the Golgi apparatus, involves several sorting stations. Since Shiga toxin receptors (Gb3) in butyric acid- treated A431 cells seem to have a ceramide moiety with longer fatty acids than in untreated cells, the possibility exists that fatty acid composition of the receptor is important for sorting to the ER. Both retrograde transport and intoxication with Shiga toxin can also be induced by cAMP, supporting the idea that retrograde transport from the Golgi to the ER is required for intoxication. The data suggest that transport to the ER in cells in situ may depend on fatty acid composition and is regulated by physiological signals.     

Regulating the actin cytoskeleton during vesicular transport

Although the actin cytoskeleton is widely believed to play an important role in intracellular protein transport, this role is poorly understood. Recently, progress has been made toward identifying specific actin-binding proteins and signaling molecules involved in regulating actin structures that function in the secretory pathway. Studies on coat protomer I (COPI)-mediated transport at the Golgi apparatus and on clathrin-mediated endocytosis have been particularly informative in identifying such mechanisms. Important similarities between actin regulation at the Golgi and at the plasma membrane have been uncovered. The studies reveal that ADP-ribosylation factor and vesicle coat proteins are able to act through the Rho-family GTP-binding proteins, Cdc42 and Rac, and several specific actin-binding proteins to direct actin assembly through the Arp2/3 complex. Efficient function of the secretory pathway is likely to require precise temporal regulation among transport-vesicle assembly, vesicle scission, and the targeting machinery. It is proposed that numerous actin regulatory mechanisms and the connections between actin signaling and vesicle-coat formation are employed to provide such temporal regulation.     

Transport of influenza virus neuraminidase (NA) to host cell surface is regulated by ARHGAP21 and Cdc42 proteins

Influenza virus neuraminidase (NA) is transported to the virus assembly site at the plasma membrane and is a major viral envelope component that plays a critical role in the release of progeny virions and in determination of host range restriction. However, little is known about the host factors that are involved in regulating the intracellular and cell surface transport of NA. Here we identified the Cdc42-specific GAP, ARHGAP21 differentially expressed in host cells infected with influenza A virus using cDNA microarray analysis. Furthermore, we have investigated the involvement of Rho family GTPases in NA transport to the cell surface. We found that expression of constitutively active or inactive mutants of RhoA or Rac1 did not significantly affect the amount of NA that reached the cell surface. However, expression of constitutively active Cdc42 or depletion of ARHGAP21 promoted the transport of NA to the plasma membranes. By contrast, cells expressing shRNA targeting Cdc42 or overexpressing ARHGAP21 exhibited a significant decrease in the amount of cell surface-localized NA. Importantly, silencing Cdc42 reduced influenza A virus replication, whereas silencing ARHGAP21 increased the virus replication. Together, our results reveal that ARHGAP21- and Cdc42-based signaling regulates the NA transport and thereby impacts virus replication.     

Functionally different pools of Shiga toxin receptor, globotriaosyl ceramide, in HeLa cells

Many studies have investigated the intracellular trafficking of Shiga toxin, but very little is known about the underlying dynamics of its cellular receptor, the glycosphingolipid globotriaosyl ceramide. In this study, we show that globotriaosyl ceramide is required not only for Shiga toxin binding to cells, but also for its intracellular trafficking. Shiga toxin induces globotriaosyl ceramide recruitment to detergent-resistant membranes, and subsequent internalization of the lipid. The globotriaosyl ceramide pool at the plasma membrane is then replenished from internal stores. Whereas endocytosis is not affected in the recovery condition, retrograde transport of Shiga toxin to the Golgi apparatus and the endoplasmic reticulum is strongly inhibited. This effect is specific, as cholera toxin trafficking on GM(1) and protein biosynthesis are not impaired. The differential behavior of both toxins is also paralleled by the selective loss of Shiga toxin association with detergent-resistant membranes in the recovery condition, and comparison of the molecular species composition of plasma membrane globotriaosyl ceramide indicates subtle changes in favor of unsaturated fatty acids. In conclusion, this study demonstrates the dynamic behavior of globotriaosyl ceramide at the plasma membrane and suggests that globotriaosyl ceramide-specific determinants, possibly its molecular species composition, are selectively required for efficient retrograde sorting on endosomes, but not for endocytosis.     

Retrograde transport of protein toxins through the Golgi apparatus

A number of protein toxins from plants and bacteria take advantage of transport through the Golgi apparatus to gain entry into the cytosol where they exert their action. These toxins include the plant toxin ricin, the bacterial Shiga toxins, and cholera toxin. Such toxins bind to lipids or proteins at the cell surface, and they are endocytosed both by clathrin-dependent and clathrin-independent mechanisms. Sorting to the Golgi and retrograde transport to the endoplasmic reticulum (ER) are common to these toxins, but the exact mechanisms turn out to be toxin and cell-type dependent. In the ER, the enzymatically active part is released and then transported into the cytosol, exploiting components of the ER-associated degradation system. In this review, we will discuss transport of different protein toxins, but we will focus on factors involved in entry and sorting of ricin and Shiga toxin into and through the Golgi apparatus.     

Targeting of Shiga toxin B-subunit to retrograde transport route in association with detergent-resistant membranes

In HeLa cells, Shiga toxin B-subunit is transported from the plasma membrane to the endoplasmic reticulum, via early endosomes and the Golgi apparatus, circumventing the late endocytic pathway. We describe here that in cells derived from human monocytes, i.e., macrophages and dendritic cells, the B-subunit was internalized in a receptor-dependent manner, but retrograde transport to the biosynthetic/secretory pathway did not occur and part of the internalized protein was degraded in lysosomes. These differences correlated with the observation that the B-subunit associated with Triton X-100-resistant membranes in HeLa cells, but not in monocyte-derived cells, suggesting that retrograde targeting to the biosynthetic/secretory pathway required association with specialized microdomains of biological membranes. In agreement with this hypothesis we found that in HeLa cells, the B-subunit resisted extraction by Triton X-100 until its arrival in the target compartments of the retrograde pathway, i.e., the Golgi apparatus and the endoplasmic reticulum. Furthermore, destabilization of Triton X-100-resistant membranes by cholesterol extraction potently inhibited B-subunit transport from early endosomes to the trans-Golgi network, whereas under the same conditions, recycling of transferrin was not affected. Our data thus provide first evidence for a role of lipid asymmetry in membrane sorting at the interface between early endosomes and the trans-Golgi network.     

Rab6 coordinates a novel Golgi to ER retrograde transport pathway in live cells

We visualized a fluorescent-protein (FP) fusion to Rab6, a Golgi-associated GTPase, in conjunction with fluorescent secretory pathway markers. FP-Rab6 defined highly dynamic transport carriers (TCs) translocating from the Golgi to the cell periphery. FP-Rab6 TCs specifically accumulated a retrograde cargo, the wild-type Shiga toxin B-fragment (STB), during STB transport from the Golgi to the endoplasmic reticulum (ER). FP-Rab6 TCs associated intimately with the ER, and STB entered the ER via specialized peripheral regions that accumulated FP-Rab6. Microinjection of antibodies that block coatomer protein I (COPI) function inhibited trafficking of a KDEL-receptor FP-fusion, but not FP-Rab6. Additionally, markers of COPI-dependent recycling were excluded from FP-Rab6/STB TCs. Overexpression of Rab6:GDP (T27N mutant) using T7 vaccinia inhibited toxicity of Shiga holotoxin, but did not alter STB transport to the Golgi or Golgi morphology. Taken together, our results indicate Rab6 regulates a novel Golgi to ER transport pathway.     

Sorting nexin 8 regulates endosome-to-Golgi transport

Sorting nexin 8 (SNX8) belongs to the sorting nexin protein family, whose members are involved in endocytosis and endosomal sorting and signaling. The function of SNX8 has so far been unknown. Here, we have investigated the role of SNX8 in intracellular transport of the bacterial toxin Shiga toxin (Stx) and the plant toxin ricin. After being endocytosed, these toxins are transported retrogradely from endosomes, via the Golgi apparatus and the endoplasmic reticulum (ER), into the cytosol, where they exert their toxic effect. Interestingly, our experiments show that SNX8 regulates the transport of Stx and ricin differently; siRNA-mediated knockdown of SNX8 significantly increased the Stx transport to the trans-Golgi network (TGN), whereas ricin transport was slightly inhibited. We also found that SNX8 colocalizes with early endosome antigen 1 (EEA1) and with retromer components, suggesting an endosomal localization of SNX8 and supporting our finding that SNX8 is involved in endosomal sorting.     

Evidence for a COP-I-independent transport route from the Golgi complex to the endoplasmic reticulum

The cytosolic coat-protein complex COP-I interacts with cytoplasmic 'retrieval' signals present in membrane proteins that cycle between the endoplasmic reticulum (ER) and the Golgi complex, and is required for both anterograde and retrograde transport in the secretory pathway. Here we study the role of COP-I in Golgi-to-ER transport of several distinct marker molecules. Microinjection of anti-COP-I antibodies inhibits retrieval of the lectin-like molecule ERGIC-53 and of the KDEL receptor from the Golgi to the ER. Transport to the ER of protein toxins, which contain a sequence that is recognized by the KDEL receptor, is also inhibited. In contrast, microinjection of anti-COP-I antibodies or expression of a GTP-restricted Arf-1 mutant does not interfere with Golgi-to-ER transport of Shiga toxin/Shiga-like toxin-1 or with the apparent recycling to the ER of Golgi-resident glycosylation enzymes. Overexpression of a GDP-restricted mutant of Rab6 blocks transport to the ER of Shiga toxin/Shiga-like toxin-1 and glycosylation enzymes, but not of ERGIC-53, the KDEL receptor or KDEL-containing toxins. These data indicate the existence of at least two distinct pathways for Golgi-to-ER transport, one COP-I dependent and the other COP-I independent. The COP-I-independent pathway is specifically regulated by Rab6 and is used by Golgi glycosylation enzymes and Shiga toxin/Shiga-like toxin-1.     

Golgi-localized GAP for Cdc42 functions downstream of ARF1 to control Arp2/3 complex and F-actin dynamics

The small GTP-binding ADP-ribosylation factor 1 (ARF1) acts as a master regulator of Golgi structure and function through the recruitment and activation of various downstream effectors. It has been proposed that members of the Rho family of small GTPases also control Golgi function in coordination with ARF1, possibly through the regulation of Arp2/3 complex and actin polymerization on Golgi membranes. Here, we identify ARHGAP10--a novel Rho GTPase-activating protein (Rho-GAP) that is recruited to Golgi membranes through binding to GTP-ARF1. We show that ARHGAP10 functions preferentially as a GAP for Cdc42 and regulates the Arp2/3 complex and F-actin dynamics at the Golgi through the control of Cdc42 activity. Our results establish a role for ARHGAP10 in Golgi structure and function at the crossroads between ARF1 and Cdc42 signalling pathways.     

Membrane trafficking at the ER/Golgi interface: functional implications of RhoA and Rac1

N-WASP and Arp2/3, the components of the actin nucleation/polymerization signaling pathway governed by Cdc42, are located in Golgi membranes and regulate ER/Golgi interface protein transport. In the present study, we examined whether RhoA and Rac1, like Cdc42, are also involved in this early secretory pathway. Unlike Cdc42, RhoA and Rac1 were not observed in the Golgi complex of different clonal cell lines nor were they present in isolated Golgi membranes. Expression of constitutively active or inactive mutants of RhoA or Rac1 proteins in HeLa cells did not alter either the disassembly or the assembly of the Golgi complex following the addition or withdrawal of BFA, respectively, the ER-to-Golgi VSV-G transport or the Sar1(dn)-induced ER accumulation of Golgi proteins. Moreover, unlike Cdc42-expressing cells, the 15 degrees C-induced subcellular redistribution of the KDEL receptor remained unaltered. Only cells that constitutively express the activated Cdc42 mutant (Cdc42Q61L), or that were microinjected with activated Cdc42Q61L protein, exhibited a significant change in Golgi complex morphology. Collectively, our results demonstrate that RhoA and Rac1 are not located in the Golgi complex, nor do they directly or indirectly regulate membrane trafficking at the ER/Golgi interface. This finding, in turn, confirms that Cdc42 is the only Rho GTPase to have a specific function on the Golgi complex.     

Endocytosis and intracellular transport of the glycolipid-binding ligand Shiga toxin in polarized MDCK cells

The glycolipid-binding cytotoxin produced by Shigella dysenteriae 1, Shiga toxin, binds to MDCK cells (strain 1) only after treatment with short-chain fatty acids like butyric acid or with the tumor promoter 12-O-tetradecanoylphorbol 13-acetate. The induced binding sites were found to be functional with respect to endocytosis and translocation of toxin to the cytosol. Glycolipids that bind Shiga toxin appeared at both the apical and the basolateral surface of polarized MDCK cells grown on filters, and Shiga toxin was found to be endocytosed from both sides of the cells. This was demonstrated by EM of cells incubated with Shiga-HRP and by subcellular fractionation of cells incubated with 125I-labeled Shiga toxin. The data indicated that toxin molecules are endocytosed from coated pits, and that some internalized Shiga toxin is transported to the Golgi apparatus. Fractionation of polarized cells incubated with 125I-Shiga toxin showed that the transport of toxin to the Golgi apparatus was equally efficient from both poles of the cells. After 1-h incubation at 37 degrees C approximately 10% of the internalized toxin was found in the Golgi fractions. The results thus suggest that glycolipids can be efficiently transported to the Golgi apparatus from both sides of polarized MDCK cell monolayers.