Actin Microfilaments Facilitate the Retrograde Transport from the Golgi Complex to the Endoplasmic Reticulum in Mammalian Cells

Abstract

The morphology and subcellular positioning of the Golgi complex depend on both microtubule and actin cytoskeletons. In contrast to microtubules, the role of actin cytoskeleton in the secretory pathway in mammalian cells has not been clearly established. Using cytochalasin D, we have previously shown that microfilaments are not involved in the endoplasmic reticulum-Golgi membrane dynamics. However, it has been reported that, unlike botulinum C2 toxin and latrunculins, cytochalasin D does not produce net depolymerization of actin filaments. Therefore, we have reassessed the functional role of actin microfilaments in the early steps of the biosynthetic pathway using C2 toxin and latrunculin B. The anterograde endoplasmic reticulum-to-Golgi transport monitored with the vesicular stomatitis virus-G protein remained unaltered in cells treated with cytochalasin D, latrunculin B or C2 toxin. Conversely, the brefeldin A-induced Golgi membrane fusion into the endoplasmic reticulum, the Golgito-endoplasmic reticulum transport of a Shiga toxin mutant form, and the subcellular distribution of the KDEL receptor were all impaired when actin microfilaments were depolymerized by latrunculin B or C2 toxin. These findings, together with the fact that COPIcoated and uncoated vesicles contain b/g-actin isoforms, indicate that actin microfilaments are involved in the endoplasmic reticulum/Golgi interface, facilitating the retrograde Golgi-to-endoplasmic reticulum membrane transport, which could be mediated by the orchestrated movement of transport intermediates along microtubule and microfilament tracks.

Actin filaments are involved in the maintenance of Golgi cisternae morphology and intra‐Golgi pH

“…Depolymerization of actin filaments and the subsequent osmotic swelling may also increase membrane tension in the Golgi complex [Morris and Homann, 2001;Sheetz, 2001] and thus decrease the reported differences in tension between the ER and Golgi [Upadhyaya and Sheetz, 2004] slowing down Golgi-to-ER membrane flow. Consistent with this hypothesis is the observation that LtB and C2 toxin treatments reduce the rate of the Golgi disassembly induced by brefeldin A [Valderrama et al, 2001]. Secondly, cisternae swelling could be also generated by the functional uncoupling of actin filaments with ion exchangers and/or vacuolar H þ -ATPases ((V)H þ -ATPases).…”

“…We have previously reported that the disruption of actin filaments causes compaction of the Golgi complex in NRK cells [Valderrama et al, 1998[Valderrama et al, , 2001. However, it is not known (i) whether this unusual Golgi morphology is generated in other mammalian cell lines when the actin cytoskeleton is disrupted by actin toxins and (ii) whether different toxins cause equivalent alterations of Golgi ultrastructure.…”

“…These disparate results are due to the fact that, unlike LtB and C2 toxin, CyD does not appear to produce a significant net depolymerization of F-actin [Morris and Tannenbaum, 1980]. However, when examining the effect of actin toxins on Golgi morphology, these three F-actin disrupters (CyD, LtB and C2 toxin) caused the same compaction of the Golgi complex [Valderrama et al, 2001], which occurred before the rounding up of cells [Valderrama et al, 1998[Valderrama et al, , 2001. Thus, results obtained with these three F-actin depolimerizing agents indicate that there is a high sensitivity of the Golgi shape to changes in the organization of the actin cytoskeleton but it is not always necessarily followed by alterations in the Golgi-associated membrane dynamics or protein transport [di Campli et al, 1999].…”

“…For instance, on the basis of results obtained using cytochalasin D (CyD) we initially reported that membrane dynamics at the ER/Golgi interface was actin-independent [Valderrama et al, 1998]. In contrast, in a subsequent study, using latrunculin B (LtB) or Clostridium botulinum C2 toxin (C2 toxin), we reported that microfilaments were involved in the retrograde (Golgi-to-ER) but not anterograde (ER-to-Golgi) membrane pathway [Valderrama et al, 2001]. These disparate results are due to the fact that, unlike LtB and C2 toxin, CyD does not appear to produce a significant net depolymerization of F-actin [Morris and Tannenbaum, 1980].…”

C3 exoenzymes, novel insights into structure and action of Rho-ADP-ribosylating toxins

“…The N-terminal part (C2IN) of C2I, which interacts with the binding component C2II, was fused to full length C3lim or C3stau respectively (Haug et al 2006;Barth et al 1998). All mammalian cells studied so far are sensitive towards the C2-C3 fusion toxin (e.g., CHO, Hela and NIH-3T3 cells; Valderrama et al 2001;Wahl et al 2000;Meyer et al 2000;Vischer et al 2000).…”