βIII Spectrin Regulates the Structural Integrity and the Secretory Protein Transport of the Golgi Complex

A spectrin-based cytoskeleton is associated with endomembranes, including the Golgi complex and cytoplasmic vesicles, but its role remains poorly understood. Using new generated antibodies to specific peptide sequences of the human βIII spectrin, we here show its distribution in the Golgi complex, where it is enriched in the trans-Golgi and trans-Golgi network. The use of a drug-inducible enzymatic assay that depletes the Golgi-associated pool of PI4P as well as the expression of PH domains of Golgi proteins that specifically recognize this phosphoinositide both displaced βIII spectrin from the Golgi. However, the interference with actin dynamics using actin toxins did not affect the localization of βIII spectrin to Golgi membranes. Depletion of βIII spectrin using siRNA technology and the microinjection of anti-βIII spectrin antibodies into the cytoplasm lead to the fragmentation of the Golgi. At ultrastructural level, Golgi fragments showed swollen distal Golgi cisternae and vesicular structures. Using a variety of protein transport assays, we show that the endoplasmic reticulum-to-Golgi and post-Golgi protein transports were impaired in βIII spectrin-depleted cells. However, the internalization of the Shiga toxin subunit B to the endoplasmic reticulum was unaffected. We state that βIII spectrin constitutes a major skeletal component of distal Golgi compartments, where it is necessary to maintain its structural integrity and secretory activity, and unlike actin, PI4P appears to be highly relevant for the association of βIII spectrin the Golgi complex.     

Morphological and biochemical analysis of the secretory pathway in melanoma cells with distinct metastatic potential.

In this report, we have investigated whether alterations of the morphological and functional aspects of the biosecretory membrane system are associated with the metastatic potential of tumor cells. To this end, we have analyzed the morphology of the Golgi complex, the cytoskeleton organization and membrane trafficking steps of the secretory pathway in two human melanoma A375 cell line variants with low (A375-P) and high metastatic (A375-MM) potential. Immunofluorescence analysis showed that in A375-P cells, the Golgi complex showed a collapsed morphology. Conversely, in A375-MM cells, the Golgi complex presented a reticular and extended morphology. At the ultrastructural level, the Golgi complex of A375-P cells was fragmented and cisternae were swollen. When the cytoskeleton was analyzed, the microtubular network appeared normal in both cell variants, whereas actin stress fibers were largely absent in A375-P, but not in A375-MM cells. In addition, the F-actin content in A375-P cells was significantly lower than in A375-MM cells. These morphological differences in A375-P cells were accompanied by acceleration and an increase in the endoplasmic reticulum to Golgi and the trans-Golgi network to cell surface membrane transport, respectively. Our results indicate that in human A375 melanoma cells, metastatic potential correlates with a well-structured morphofunctional organization of the Golgi complex and actin cytoskeleton.     

Poliovirus infection and expression of the poliovirus protein 2B provoke the disassembly of the Golgi complex, the organelle target for the antipoliovirus drug Ro-090179.

Infection of Vero cells with poliovirus results in complete disassembly of the Golgi complex. Milestones of the process of disassembly are the release to the cytosol of the beta-COP bound to Golgi membranes, the disruption of the cis-Golgi network into fragments scattered throughout the cytoplasm, and the disassembly of the stacked cisternae by a process mediated by long tubular structures. Transient expression of the viral protein 2B in COS-7 cells also causes the disassembly of the Golgi complex by a process preceded by the accumulation of the protein in the Golgi area. Vero cells infected for 3 h show no recognizable Golgi complexes at the ultrastructural level and display an enormously swollen endoplasmic reticulum (ER) with extensive areas of its surface heavily coated. Ro-090179 (Ro), a flavonoid isolated from the herb Agastache rugosa, provokes the specific swelling and disruption of the Golgi complex and strongly inhibits poliovirus infection. Ro provokes the swelling and the disruption of the stacked cisternae and trans-Golgi elements without affecting the cis-most Golgi cisternae much. Moreover, Ro inhibits the fusion of the Golgi complex with the ER in cells treated with brefeldin A and provokes the accumulation of the intermediate compartment membrane protein p58 into ERD2-positive Golgi elements but has no effect on the anterograde transport involved in protein secretion. Our results indicate that the secretory pathway and specifically the Golgi complex are preferential targets of poliovirus.     

Arginine-vasopressin-induced structural alterations in MDCK cells

Alterations in the structural organization of MDCK cells under the effect of arginine-vasopressin (AVP) have been studied using electron and fluorescent microscopy methods. Electron microscopy has confirmed that the MDCK cells in the monolayer have structurally different apical and basolateral surfaces separated by well-formed zones of intercellular contacts. AVP has been proven to bind specifically to receptors on the basolateral cell surface and be internalized from the cell surface after 10–15 min. AVP produces fragmentation of the Golgi apparatus and swelling in its cisternae due to the appearance of an osmotic water flow across the monolayer. The significant depolymerization of the cell’s actin cytoskeleton has been revealed under effect of AVP or forskolin (an adenylyl cyclase activator). The functional role and regulatory mechanisms of the described structural alterations are discussed.     

Association of Cdc42/N-WASP/Arp2/3 signaling pathway with Golgi membranes

Recent findings indicate that Cdc42 regulates Golgi-to-ER (endoplasmic reticulum) protein transport through N-WASP and Arp2/3 (Luna et al. 2002, Mol. Biol. Cell, 13:866-879). To analyse the components of the Cdc42-governed signaling pathway in the secretory pathway, we localized Cdc42, N-WASP and Arp2/3 in the Golgi complex by cryoimmunoelectron microscopy. Cdc42 is found throughout the Golgi stack, particularly in cis/middle cisternae, whereas N-WASP and Arp3 (a component of the Arp2/3 complex) are restricted to cis cisternae. Arp3 also colocalized in peri-Golgi tubulovesicular structures with either KDEL receptor or GM130. Even though Arp3 is not found in TGN46-positive cisternal elements, a small fraction of Arp3-labeled tubulo-vesicular elements showed TGN46 labeling. Active Cdc42 (GTP-bound form) induced relocation of N-WASP and Arp3 to the lateral rims of Golgi cisternae. These results show that the actin nucleation and polymerization signaling pathway governed by Cdc42/N-WASP/Arp operates in the Golgi complex of mammalian cells, further implicating actin dynamics in Golgi-associated membrane trafficking.     

Actin depolymerizing factor stabilizes an existing state of F-actin and can change the tilt of F-actin subunits

Proteins in the actin depolymerizing factor (ADF)/cofilin family are essential for rapid F-actin turnover, and most depolymerize actin in a pH-dependent manner. Complexes of human and plant ADF with F-actin at different pH were examined using electron microscopy and a novel method of image analysis for helical filaments. Although ADF changes the mean twist of actin, we show that it does this by stabilizing a preexisting F-actin angular conformation. In addition, ADF induces a large ( approximately 12 degrees ) tilt of actin subunits at high pH where filaments are readily disrupted. A second ADF molecule binds to a site on the opposite side of F-actin from that of the previously described ADF binding site, and this second site is only largely occupied at high pH. All of these states display a high degree of cooperativity that appears to be an integral part of F-actin.     

Involvement of the Rho-mDia1 pathway in the regulation of Golgi complex architecture and dynamics

In mammalian cells, the Golgi apparatus is a ribbon-like, compact structure composed of multiple membrane stacks connected by tubular bridges. Microtubules are known to be important to Golgi integrity, but the role of the actin cytoskeleton in the maintenance of Golgi architecture remains unclear. Here we show that an increase in Rho activity, either by treatment of cells with lysophosphatidic acid or by expression of constitutively active mutants, resulted in pronounced fragmentation of the Golgi complex into ministacks. Golgi dispersion required the involvement of mDia1 formin, a downstream target of Rho and a potent activator of actin polymerization; moreover, constitutively active mDia1, in and of itself, was sufficient for Golgi dispersion. The dispersion process was accompanied by formation of dynamic F-actin patches in the Golgi area. Experiments with cytoskeletal inhibitors (e.g., latrunculin B, blebbistatin, and Taxol) revealed that actin polymerization, myosin-II-driven contractility, and microtubule-based intracellular movement were all involved in the process of Golgi dispersion induced by Rho-mDia1 activation. Live imaging of Golgi recovery revealed that fusion of the small Golgi stacks into larger compartments was repressed in cells with active mDia1. Furthermore, the formation of Rab6-positive transport vesicles derived from the Golgi complex was enhanced upon activation of the Rho-mDia1 pathway. Transient localization of mDia1 to Rab6-positive vesicles was detected in cells expressing active RhoA. Thus, the Rho-mDia1 pathway is involved in regulation of the Golgi structure, affecting remodeling of Golgi membranes.     

The C-terminal tail of UNC-60B (actin depolymerizing factor/cofilin) is critical for maintaining its stable association with F-actin and is implicated in the second actin-binding site

Actin depolymerizing factor (ADF)/cofilin changes the twist of actin filaments by binding two longitudinally associated actin subunits. In the absence of an atomic model of the ADF/cofilin-F-actin complex, we have identified residues in ADF/cofilin that are essential for filament binding. Here, we have characterized the C-terminal tail of UNC-60B (a nematode ADF/cofilin isoform) as a novel determinant for its association with F-actin. Removal of the C-terminal isoleucine (Ile152) by carboxypeptidase A or truncation by mutagenesis eliminated F-actin binding activity but strongly enhanced actin depolymerizing activity. Replacement of Ile152 by Ala had a similar but less marked effect; F-actin binding was weakened and depolymerizing activity slightly enhanced. Truncation of both Arg151 and Ile152 or replacement of Arg151 with Ala also abolished F-actin binding and enhanced depolymerizing activity. Loss of F-actin binding in these mutants was accompanied by loss or greatly decreased severing activity. All of the variants of UNC-60B interacted with G-actin in an indistinguishable manner from wild type. Cryoelectron microscopy showed that UNC-60B changed the twist of F-actin to a similar extent to vertebrate ADF/cofilins. Helical reconstruction and structural modeling of UNC-60B-F-actin complex reveal how the C terminus of UNC-60B might be involved in one of the two actin-binding sites.     

Endogenous and monoclonal antibodies to the rat pancreatic acinar cell Golgi complex

Normal, unimmunized mouse serum from several strains (BALB/c, C57/b, DBA/2, NZB, SJL, CD/1) contains an endogenous IgG antibody that localizes to the Golgi complex of rat pancreatic acinar cells. Treatment of pancreatic acini with 5 microM monensin resulted in the swelling and vacuolization of the Golgi cisternae, and in a corresponding annular staining by the mouse serum as observed by immunofluorescence, suggesting that the antigen recognized is on the Golgi complex cisternal membrane. The antiserum did not react with pancreatic secretory proteins, and its binding to smooth microsomal membranes was retained following sodium carbonate washing, supporting a Golgi membrane localization. Advantage was taken of the existence of the endogenous murine antibody for the isolation of monoclonal antibodies directed to the Golgi complex of the rat pancreas. Two antibodies, antiGolgi 1 and antiGolgi 2, are described. Both antibodies are IgMs that recognize integral membrane proteins of the trans-Golgi cisternae, with lighter and patchy staining of the pancreatic lumen membrane, as observed both by light and electron microscopy. AntiGolgi 1 recognizes predominately a protein of molecular weight 103,000- 108,000, whereas antiGolgi 2 shows a strong reaction to a 180-kd band as well as the 103-108-kd protein.     

pH-independent retrograde targeting of glycolipids to the Golgi complex

A small fractionof the molecules internalized by endocytosis reaches the Golgi complexthrough a retrograde pathway that is poorly understood. In the presentwork, we used bacterial toxins to study the retrograde pathway in Verocells. The recombinant B subunit of verotoxin 1B (VT1B)was labeled with fluorescein to monitor its progresswithin the cell by confocal microscopy. This toxin, which bindsspecifically to the glycolipid globotriaosyl ceramide, enteredendosomes by both clathrin-dependent and -independent pathways,reaching the Golgi complex. Once internalized, the toxin-receptor complex did not recycle back to the plasma membrane. The kinetics ofinternalization and the subcellular distribution of VT1B were virtuallyidentical to those of another glycolipid-binding toxin, the B subunitof cholera toxin (CTB). Retrograde transport of VT1B and CTB wasunaffected by addition of weak bases in combination with concanamycin,a vacuolar-type ATPase inhibitor. Ratio imaging confirmed that theseagents neutralized the luminal pH of the compartments where the toxinwas located. Therefore, the retrograde transport of glycolipids differsfrom that of proteins like furin and TGN38, which require an acidicluminal pH. Additional experiments indicated that the glycolipidreceptors of VT1B and CTB are internalized independently and not aspart of lipid "rafts" and that internalization is cytochalasininsensitive. We conclude that glycolipids utilize a unique,pH-independent retrograde pathway to reach compartments of thesecretory system and that assembly of F-actin is not required for thisprocess.

     

V-ATPase is a major component of the Golgi complex in the scaly green flagellate Scherffelia dubia

Highly purified membranes isolated from the Golgi complex of the scaly green flagellate Scherffelia dubia (Chlorophyta) were subjected to Triton X-114 two-phase partitioning. Proteins in the detergent phase were analyzed by 2D gel electrophoresis and a major protein of 66 kD (p66) was N-terminally sequenced. The complete cDNA sequence of p66 was obtained by 3' RACE-PCR and screening of a cDNA library of S. dubia with a PCR probe derived from the 3' RACE. Sequence analysis of the cDNA clone identified p66 as subunit A of V-ATPase. Other major proteins in the isolated Golgi complex were immunoreactive to heterologous antibodies raised against subunit B or the holoenzyme of V-ATPase. A polyclonal (anti-p66) antibody raised against a recombinant, bacterially expressed p66 fusion protein recognized p66 in the isolated Golgi complex in western blots and localized the antigen by immunogold electron microscopy mostly to the scale reticulum but also to the Golgi stack within the Golgi complex. Concanamycin A-sensitive (but bafilomycin A1-insensitive) ATPase activity was present in the isolated Golgi complex, and monensin at 0.5-1 microM reversibly inhibited flagellar regeneration and resulted in swelling of Golgi cisternae. It is concluded that a functional V-ATPase is a major protein of the Golgi complex in S. dubia and is presumably associated with sorting processes at the endocytotic/exocytotic boundary of the Golgi complex.     

Binding of the N-terminal fragment C0-C2 of cardiac MyBP-C to cardiac F-actin

Cardiac myosin-binding protein C (cMyBP-C), a major accessory protein of cardiac thick filaments, is thought to play a key role in the regulation of myocardial contraction. Although current models for the function of the protein focus on its binding to myosin S2, other evidence suggests that it may also bind to F-actin. We have previously shown that the N-terminal fragment C0-C2 of cardiac myosin-binding protein-C (cMyBP-C) bundles actin, providing evidence for interaction of cMyBP-C and actin. In this paper we directly examined the interaction between C0-C2 and F-actin at physiological ionic strength and pH by negative staining and electron microscopy. We incubated C0-C2 (5-30μM, in a buffer containing in mM: 180 KCl, 1 MgCl(2), 1 EDTA, 1 DTT, 20 imidazole, at pH 7.4) with F-actin (5μM) for 30min and examined negatively-stained samples of the solution by electron microscopy (EM). Examination of EM images revealed that C0-C2 bound to F-actin to form long helically-ordered complexes. Fourier transforms indicated that C0-C2 binds with the helical periodicity of actin with strong 1st and 6th layer lines. The results provide direct evidence that the N-terminus of cMyBP-C can bind to F-actin in a periodic complex. This interaction of cMyBP-C with F-actin supports the possibility that binding of cMyBP-C to F-actin may play a role in the regulation of cardiac contraction.     

The Golgi apparatus is a primary site of intracellular damage after photosensitization with Rose Bengal acetate

The aim of the present investigation was to elucidate whether the Golgi apparatus undergoes photodamage following administration of the fluorogenic substrates Rose Bengal acetate (RBAc) and irradiation at the appropriate wavelength. Human HeLa cells were treated in culture and the changes in the organization of the Golgi apparatus were studied using fluorescence confocal microscopy and electron microscopy, after immunocytochemical labeling. To see whether the cytoskeletal components primarily involved in vesicle traffic (i.e., microtubules) might also be affected, experiments of tubulin immunolabeling were performed. After treatment with RBAc and irradiation, cells were allowed to grow in drug-free medium for different times. 24 hr after irradiation, the cisternae of the Golgi apparatus became packed, and after 48-72 hr they appeared more fragmented and scattered throughout the cytoplasm; these changes in the organization of the Golgi cisternae were confirmed at electron microscopy. Interestingly enough, apoptosis was found to occur especially 48-72 h after irradiation, and apoptotic cells exhibited a dramatic fragmentation of the Golgi membranes. The immunolabeling with anti-tubulin antibody showed that microtubules were also affected by irradiation in RBAc-treated cells.     

A new model for the interaction of dystrophin with F-actin

The F-actin binding and cross-linking properties of skeletal muscle dystrophin-glycoprotein complex were examined using high and low speed cosedimentation assays, microcapillary falling ball viscometry, and electron microscopy. Dystrophin-glycoprotein complex binding to F-actin saturated near 0.042 +/- 0.005 mol/ mol, which corresponds to one dystrophin per 24 actin monomers. Dystrophin-glycoprotein complex bound to F-actin with an average apparent Kd for dystrophin of 0.5 microM. These results demonstrate that native, full-length dystrophin in the glycoprotein complex binds F-actin with some properties similar to those measured for several members of the actin cross-linking super- family of proteins. However, we failed to observe dystrophin- glycoprotein complex-induced cross-linking of F-actin by three different methods, each positively controlled with alpha-actinin. Furthermore, high speed cosedimentation analysis of dystrophin- glycoprotein complex digested with calpain revealed a novel F-actin binding site located near the middle of the dystrophin rod domain. Recombinant dystrophin fragments corresponding to the novel actin binding site and the first 246 amino acids of dystrophin both bound F- actin but with significantly lower affinity and higher capacity than was observed with purified dystrophin-glycoprotein complex. Finally, dystrophin-glycoprotein complex was observed to significantly slow the depolymerization of F-actin, Suggesting that dystrophin may lie along side an actin filament through interaction with multiple actin monomers. These data suggest that although dystrophin is most closely related to the actin cross-linking superfamily based on sequence homology, dystrophin binds F-actin in a manner more analogous to actin side-binding proteins.     

A distinct class of vesicles derived from the trans‐Golgi mediates secretion of xylogalacturonan in the root border cell

Root border cells lie on the surface of the root cap and secrete massive amounts of mucilage that contains polysaccharides and proteoglycans. Golgi stacks in the border cells have hypertrophied margins, reflecting elevated biosynthetic activity to produce the polysaccharide components of the mucilage. To investigate the three‐dimensional structures and macromolecular compositions of these Golgi stacks, we examined high‐pressure frozen/freeze‐substituted alfalfa root cap cells with electron microscopy/tomography. Golgi stacks in border cells and peripheral cells, precursor cells of border cells, displayed similar morphological features, such as proliferation of trans cisternae and swelling of the trans cisternae and trans‐Golgi network (TGN) compartments. These swollen margins give rise to two types of vesicles larger than other Golgi‐associated vesicles. Margins of trans‐Golgi cisternae accumulate the LM8 xylogalacturonan (XGA) epitope, and they become darkly stained large vesicles (LVs) after release from the Golgi. Epitopes for xyloglucan (XG), polygalacturonic acid/rhamnogalacturonan‐I (PGA/RG‐I) are detected in the trans‐most cisternae and TGN compartments. LVs produced from TGN compartments (TGN‐LVs) stained lighter than LVs and contained the cell wall polysaccharide epitopes seen in the TGN. LVs carrying the XGA epitope fuse with the plasma membrane only in border cells, whereas TGN‐LVs containing the XG and PGA/RG‐I epitopes fuse with the plasma membrane of both peripheral cells and border cells. Taken together, these results indicate that XGA is secreted by a novel type of secretory vesicles derived from trans‐Golgi cisternae. Furthermore, we simulated the collapse in the central domain of the trans‐cisternae accompanying polysaccharide synthesis with a mathematical model.     

Simultaneous visualization of the actin plate and new cell partition membrane during cytokinesis in the brown alga Sphacelaria rigidula (Sphacelariales,Phaeophyceae)

In many brown algae, cytokinesis is accomplished through the centrifugal expansion of the membrane structure formed by the fusion of Golgi vesicles and flat cisternae. In contrast, it has been reported that cytokinesis in Sphacelaria rigidula progresses centripetally by adding Golgi vesicles and flat cisternae to cleaving furrows of the plasma membrane. The reason why this cytokinetic pattern was observed only in Sphacelaria species is unknown. In either cytokinesis pattern, a plate-like actin structure (the actin plate) coincides with the cytokinetic plane between the daughter nuclei. However, it is unclear how the actin plate is related to cytokinesis progression. In this study, we re-examined cytokinesis in the apical cells of S. rigidula using transmission electron microscopy. Double staining of the actin plate and the developing membrane was followed by fluorescence microscopy analysis to determine the relationship between these two formations. The results showed that cytokinesis in S. rigidula, as in many brown algae, was completed by centrifugal growth of the new cell partition membrane. A furrow of the plasma membrane was observed at the beginning of cytokinesis; however, further invagination did not occur. The actin plate arose at the center of the cytokinetic plane before membrane fusion and extended parallel to the expansion of the new cell partition membrane. When cytokinesis was slow due to insufficient Golgi vesicle supply to the cytokinetic plane in the cells under brefeldin A treatment, the extension of the actin plate was also suspended. In this study, the spatiotemporal relationship between the occurrence and expansion of the actin plate and the new cell partition membrane was revealed. These observations indicate that the actin plate might promote membrane fusion or lead to the growth of a new cell partition membrane.     

Monensin-induced swelling of Golgi apparatus cisternae mediated by a proton gradient

Monensin, a monovalent ionophore, caused swelling of mature cisternae of plant Golgi apparatus. The appearance of swollen cisternae was time-dependent and linear over a period of 1 h with an estimated maximum rate of production of one swollen cisterna every 3 to 4 min. Implicit in these observations was a need for the uptake of osmotically active monovalent cations to have occurred accompanied by a concomitant efflux of H+ and the entry of water. Furthermore, to sustain the H+ efflux, a source of H+ influx also would be required. To test for the latter, cisternal swelling, as visualized by electron microscopy, was monitored by treatment of wild carrot cells in suspension culture with drugs and inhibitors known to interfere with proton gradients. Swelling was inhibited by the protonophore, FCCP, by the inhibitor of lysosomal acidification, quercetin, and by the lysosomotropic amines, chloroquine and ammonia. While antimycin A, an inhibitor of mitochondrial oxidative phosphorylation, was ineffective, cyanide dramatically decreased swelling. The numbers of swollen cisternae produced could be reduced by prolonged treatment with arsenate, such that an ATP requirement is indicated, at least, for cisternal formation. Swelling was promoted by citrate, representative of a permeant organic anion. Reductions in numbers of monensin-induced swollen cisternae in the presence of quercetin, vanadate, and chloroquine could be compensated for by the addition of citrate. We conclude that the monensin-induced swelling of Golgi apparatus cisternae may involve a mechanism generating a proton gradient at or near the mature Golgi apparatus face.     

The AE2 anion exchanger is necessary for the structural integrity of the Golgi apparatus in mammalian cells

The structural integrity of the Golgi apparatus is known to be dependent on multiple factors, including the organizational status of microtubules, actin and the ankyrin/spectrin-based Golgi membrane skeleton, as well as vesicular trafficking and pH homeostasis. In this respect, our recently identified Golgi-associated anion exchanger, AE2, may also be of importance, since it potentially acts as a Golgi pH regulator and as a novel membrane anchor for the spectrin-based Golgi membrane skeleton. Here, we show that inhibition (>75%) of AE2 expression by antisense oligonucleotides in COS-7 cells results in the fragmentation of the juxtanuclear Golgi apparatus and in structural disorganization of the Golgi stacks, the cisternae becoming generally shorter, distorted, vesiculated and/or swollen. These structural changes occurred without apparent dissociation of the Golgi membrane skeletal protein Ankyrin(195), but were accompanied by the disappearance of the well-focused microtubule-organizing center (MTOC), suggesting the involvement of microtubule reorganization. Similar changes in Golgi structure and assembly of the MTOC were also observed upon transient overexpression of the EGFP-AE2 fusion protein. These data implicate a clear structural role for the AE2 protein in the Golgi and in its cytological positioning around the MTOC.     

Perturbation of the secretory network inClosterium acerosum by Na+-selective ionophores

Summary Closterium acerosum possesses a well-defined, mucilage-secretory mechanism consisting of up to 100 Golgi bodies, two distinct vacuolar networks, and an active cytoplasmic-streaming network located in the cell periphery. Five different sodium-affecting agents were applied to actively secreting cells in order to determine the role, if any, of Na⁺ on this secretory mechanism. Significant effects to the endomembrane system and actin cytoskeleton were noted upon treatment with the Na⁺-specific ionophores monensin and SQl-Et. In particular, the following alterations were noted: incurling of Golgi cisternae and the formation of circular cisternal profiles at the trans face, swelling of the cis-medial cisternae, and dissociation of the Golgi body from the internal cytoplasm to the peripheral cytoplasmic zones. Immunogold labeling with a mucilage-specific polyclonal antibody reveals that mucilage production is diminished during longer ionophore treatments. Likewise, both the polar and peripheral vacuoles disintegrate into a series of smaller vacuoles. Cytoplasmic streaming ceases and the normal actin network of the peripheral cytoplasm transforms into irregularly spaced fibrillar bundles. Finally, multilaminate structures appear at the plasma membrane. No cytological effects could be observed with the Na⁺-channel blockers or Na⁺-current transducers QX-14, tetrodotoxin, or amiloride.Abbreviations DIC differential interference contrast - GA Golgi apparatus - LM light microscopy - TEM transmission electron microscopy - TGN trans Golgi network - WHM Woods Hole medium - DMSO dimethylsulfoxide     

Golgi body motility in the plant cell cortex correlates with actin cytoskeleton organization

The actin cytoskeleton is involved in the transport and positioning of Golgi bodies, but the actin-based processes that determine the positioning and motility behavior of Golgi bodies are not well understood. In this work, we have studied the relationship between Golgi body motility behavior and actin organization in intercalary growing root epidermal cells during different developmental stages. We show that in these cells two distinct actin configurations are present, depending on the developmental stage. In small cells of the early root elongation zone, fine filamentous actin (F-actin) occupies the whole cell, including the cortex. In larger cells in the late elongation zone that have almost completed cell elongation, actin filament bundles are interspersed with areas containing this fine F-actin and areas without F-actin. Golgi bodies in areas with the fine F-actin exhibit a non-directional, wiggling type of motility. Golgi bodies in areas containing actin filament bundles move up to 7 μm s?1. Since the motility of Golgi bodies changes when they enter an area with a different actin configuration, we conclude that the type of movement depends on the actin organization and not on the individual organelle. Our results show that the positioning of Golgi bodies depends on the local actin organization.