A prelysosomal compartment sequesters membrane-impermeant fluorescent dyes from the cytoplasmic matrix of J774 macrophages

After the membrane impermeant dye Lucifer Yellow is introduced into the cytoplasmic matrix of J774 cells, the dye is sequestered within cytoplasmic vacuoles and secreted into the extracellular medium. In the present work we studied the intracellular transport of Lucifer Yellow in J774 macrophages and the nature of the cytoplasmic vacuoles into which this dye is sequestered. When the lysosomal system of J774 cells was prelabeled with a Texas red ovalbumin conjugate and Lucifer Yellow was then loaded into the cytoplasm of the cells by ATP-mediated permeabilization of the plasma membrane, the vacuoles that sequestered Lucifer Yellow 30 min later were distinct from the Texas red-stained lysosomes. After an additional 30 min Lucifer Yellow and Texas red colocalized in the same membrane bound compartments, indicating that the Lucifer Yellow had been delivered to lysosomes. We next prelabeled the plasma membrane of J774 cells with anti-macrophage antibody and Texas red protein A before Lucifer Yellow was loaded into the cells. The phase-lucent vacuoles that subsequently sequestered Lucifer Yellow also stained with Texas red, showing that they were part of the endocytic pathway. J774 cells were fractionated on percoll density gradients either 15 or 60 min after Lucifer Yellow was introduced into the cytoplasmic matrix of the cells. In cells fractionated after 15 min, Lucifer Yellow was contained within the fractions of light buoyant density that contain plasma membrane and endosomes; the dye later appeared in vesicles of higher density which contained lysosomes. Secretion of Lucifer Yellow from the cytoplasmic matrix of J774 cells is inhibited by the organic anion transport blocker probenecid. We found that probenecid also reversibly inhibited sequestration of dye, indicating that sequestration of dye within cytoplasmic vacuoles was also mediated by organic anion transporters. These studies show that the vacuoles that sequester Lucifer Yellow from the cytoplasmic matrix of J774 cells possess the attributes of endosomes. Thus, in addition to their role in sorting of membrane bound and soluble substances, macrophage endosomes may play a role in the accumulation and transport of molecules resident in the soluble cytoplasm.     

Macrophages possess probenecid-inhibitable organic anion transporters that remove fluorescent dyes from the cytoplasmic matrix

We introduced several membrane-impermeant fluorescent dyes, including Lucifer Yellow, carboxyfluorescein, and fura-2, into the cytoplasmic matrix of J774 cells and thioglycollate-elicited mouse peritoneal macrophages by ATP permeabilization of the plasma membrane and observed the subsequent fate of these dyes. The dyes did not remain within the cytoplasmic matrix; instead they were sequestered within phase-lucent cytoplasmic vacuoles and released into the extracellular medium. We used Lucifer Yellow to study these processes further. In cells incubated at 37 degrees C, 87% of Lucifer Yellow was released from the cells within 30 min after dye loading. The dye that remained within the cells at this time was predominantly within cytoplasmic vacuoles. Lucifer yellow transport was temperature dependent and occurred against a concentration gradient; therefore it appeared to be an energy- requiring process. The fluorescent dyes used in these studies are all organic anions. We therefore examined the ability of probenecid (p- [dipropylsulfamoyl]benzoic acid), which blocks organic anion transport across many epithelia, to inhibit efflux of Lucifer Yellow, and found that this drug inhibited this process in a dose-dependent and reversible manner. Efflux of Lucifer Yellow from the cells did not require Na+ co-transport or Cl- antiport; however, it was inhibited by lowering of the extracellular pH. These experiments indicate that macrophages possess probenecid-inhibitable transporters which are similar in their functional properties to organic anion transporters of epithelial cells. Such organic anion transporters have not been described previously in macrophages; they may mediate the release of naturally occurring organic anions such as prostaglandins, leukotrienes, glutathione, bilirubin, or lactate from macrophages.     

Inhibition of Fura-2 sequestration and secretion with organic anion transport blockers

Fura-2 is widely used to measure the concentration of cytosolic free calcium, but in many cells the dye does not remain localized within the cytoplasmic matrix. In these cells, Fura-2 is sequestered within intracellular organelles, secreted into the extracellular medium, or both. We have found that, in mouse peritoneal macrophages, J774 cells, PC12 cells, and N2A cells, Fura-2 sequestration and secretion are mediated by organic anion transport systems and are blocked by the inhibitors probenecid and sulfinpyrazone. Under appropriate conditions these agents have little affect on calcium transients, and may facilitate the use of Fura-2 in a variety of cell types.     

Direct observation of rapid internalization and intracellular transport of sterol by macrophage foam cells

Transport of the fluorescent cholesterol analog dehydroergosterol (DHE) from the plasma membrane was studied in J774 macrophages (Mphis) with normal and elevated cholesterol content. Cells were labeled with DHE bound to methyl-beta-cyclodextrin. In J774, Mphis with normal cholesterol, intracellular DHE became enriched in recycling endosomes, but was not highly concentrated in the trans-Golgi network or late endosomes and lysosomes. After raising cellular cholesterol by incubation with acetylated low-density lipoprotein (AcLDL), DHE was transported to lipid droplets, and less sterol was found in recycling endosomes. Transport of DHE to droplets was very rapid (t1/2 = 1.5 min after photobleaching) and did not require metabolic energy. In cholesterol-loaded J774 Mphis, the initial fraction of DHE in the plasma membrane was reduced, and rapid DHE efflux from the plasma membrane to intracellular organelles was observed. This rapid sterol transport was not related to plasma membrane vesiculation, as DHE did not become enriched in endocytic vesicles formed after sphingomyelinase C treatment of cells. When cells were incubated with DHE ester incorporated into AcLDL, fluorescence of the sterol was first found in punctate endosomes. After a chase, this DHE colocalized with transferrin in a distribution similar to cells labeled with DHE delivered by methyl-beta-cyclodextrin. Our results indicate that elevation of sterol levels in Mphis enhances transport of sterol from the plasma membrane by a non-vesicular pathway.     

Fura-2 secretion and sequestration in macrophages. A blocker of organic anion transport reveals that these processes occur via a membrane transport system for organic anions

Fura-2, loaded into J774.2 macrophages as the acetoxymethyl ester, is sequestered into intracellular vacuoles within 90 min after the beginning of the loading at 37 degrees C. The dye is also efficiently secreted from the cells. Sequestration and secretion of fura-2 reduce the accuracy of measurements of cytosolic free Ca2+ concentration in this cell line. Fura-2 is also sequestered and secreted by J774.2 when the dye is loaded into the cytoplasm as the pentapotassium salt by reversible permeabilization of the plasma membrane. Regardless of the mechanism by which fura-2 is loaded into the cytoplasm, both sequestration and secretion are prevented by 2.5 mM probenecid, a blocker of organic anion transport. Probenecid has no effect on resting or stimulated cytosolic free Ca2+ levels or on FcR-mediated phagocytosis. These findings suggest that macrophages express a transport mechanism for the anionic form of fura-2. This transport system is responsible for the clearance of fura-2 from the cytoplasm of this cell type. Furthermore we suggest that use of probenecid to block secretion and intracellular sequestration of fura-2 may overcome problems arising in the application of this Ca2+ indicator to macrophages and perhaps to other cell types.     

Vacuolar Lucifer Yellow Uptake in Plants: Endocytosis or Anion Transport; A Critical Opinion

Because of its membrane-impermeant-properties Lucifer Yellow-CH (LY) is regarded by animal cell biologists as an ideal tracer for fluid-phase endocytosis. When presented to plant cells or protoplasts this fluoroprobe accumulates in the vacuole. On the other hand there are many cases where LY does not enter the vacuole when loaded into the plant cytosol. These, superficially divergent, results have previously been explained in terms of endocytosis whereby access to the vacuole is considered to occur through vesicle transport. This interpretation has now been challenged in three recent papers where the benzoic acid derivative, probenecid, has been shown to prevent vacuolar LY accumulation in plants. Since probenecid is a well-known inhibitor of organic anion transport in animal cells it has been argued that anion carriers capable of transporting LY might also exist at the plasma membrane and tonoplast of plant cells. Unfortunately probenecid has rarely, if ever, been used in plant transport studies. The fact that it is a weak acid, whose inhibitory effects are observed at concentrations of around 1 mM suggests that caution should prevail when interpreting results obtained with probenecid. The purpose of this article is therefore to highlight the current controversy surrounding LY uptake by plants and to critically evaluate the recent probenecid data.     

Physicochemical properties alone do not predict the movement and compartmentation of fluorescent xenobiotics

Oat aleurone protoplasts, maintained in sterile liquid culturefor 5 d, are able to take up a number of fluorescent probesof varying charge and of molecular weights in the range 457to 637. In addition to Lucifer Yellow CH, these include PTS,HPTS, Lucifer Yellow AB, calcein, and sulphorhodamine-101, mostof which have previously been described as membrane-impermeantdue to their physicochemical properties. The transport of theseprobes across the plasma membrane and their subsequent sequestrationwithin the vacuole, is inhibited by the drug probenecid, negatingthe possibility that movement is solely by simple diffusion.In contrast, Trypan blue (mol. wt. 961) is excluded by all liveoat aleurone protoplasts. The uptake of carboxyfluorescein into protoplasts during theearly stages of development can, in part, be explained by diffusionof the undissociated molecule and subsequent anion trappingin the cytosol. However, both the uptake into the protein bodiesof 1-d-old protoplasts and into the vacuoles of 5-d-old protoplastsis inhibited by probenecid. This indicates that the transportof carboxyfluorescein is carrier-mediated and that the carrieris present on the tono-plast membrane throughout protoplastdevelopment. Since probes such as carboxyfluorescein have physicochemicalproperties similar to some phloem-mobile xenobiotics, the resultshave important implications for theories pertaining to the movementand compartmentation of xenobiotics within plants. Key words: Aleurone protoplast, oat (Avena sativa), fluorescent xenobiotics, probenecid, transport     

Uptake and compartmentalisation of fluorescent probes byPisolithus tinctorius hyphae: evidence for an anion transport mechanism at the tonoplast but not for fluid-phase endocytosis

Summary Membrane-impermeant fluorescent probes, such as Lucifer Yellow carbohydrazide, 6-carboxyfluorescein, and high-molecular-mass fluorescent dextrans (10 and 70 kDa) are not internalised by actively-growing hyphal tip-cells ofPisolithus tinctorius even after prolonged exposure to the probe. These findings suggest that fluid-phase endocytosis may not occur in these fully turgid tip-growing hyphae. In contrast, a number of membrane-permeant fluorescent probes, including 6-carboxfluorescein diacetate, the novel fluorescein-substitute Oregon Green 488 carboxylic acid diacetate, and the thiol-reactive Cell Tracker reagents 7-amino-4-chloro-methylcoumarin and 5-chloromethylfluorescein diacetate, are taken up by these hyphae and their fluorescent products accumulate in the vacuole system. Accumulation of the fluorescent products of both 6-carboxyfluorescein diacetate and Oregon Green 488 carboxylic acid diacetate in the vacuole system is inhibited by the anion transport inhibitor probenecid and instead these fluorochromes remain in the cytoplasm. These results suggest that the membrane-permeant esters 6-carboxyfluorescein diacetate and Oregon Green 488 carboxylic acid diacetate are first hydrolysed in the cytoplasm and that their fluorescent products are subsequently sequestered across the tonoplast via an anion transport mechanism. Such an anion transport mechanism has been hitherto unrecognised in fungi and may serve to detoxify the fungal cytoplasm by the removal of naturally-occurring unwanted anions. Probenecid-inhibitable organic anion transporters are also located at the limiting membrane of the animal endosomal/lysosomal system and at the tonoplast of higher plants. Our results further support the idea that the tubular vacuole system inP. tinctorius is similar to animal endosomal/lysosomal and plant vacuole systems.     

Inhibition of organic anion transport in endothelial cells by hydrogen peroxide.

ATP loss is a prominent feature of cellular injury induced by oxidants or ischemia. How reduction of cellular ATP levels contributes to lethal injury is still poorly understood. In this study we examined the ability of H2O2 to inhibit in a dose-dependent manner the extrusion of fluorescent organic anions from bovine pulmonary artery endothelial cells. Extrusion of fluorescent organic anions was inhibited by probenecid, suggesting an organic anion transporter was involved. In experiments in which ATP levels in endothelial cells were varied by treatment with different degrees of metabolic inhibition, it was determined that organic anion transport was ATP-dependent. H2O2-induced inhibition of organic anion transport correlated well with the oxidant's effect on cellular ATP levels. Thus H2O2-mediated inhibition of organic anion transport appears to be via depletion of ATP, a required substrate for the transport reaction. Inhibition of organic anion transport directly by probenecid or indirectly by metabolic inhibition with reduction of cellular ATP levels was correlated with similar reductions of short term viability. This supports the hypothesis that inhibition of organic anion transport after oxidant exposure or during ischemia results from depletion of ATP and may significantly contribute to cytotoxicity.     

The endosomal proteome of macrophage and dendritic cells

The essential roles of the endovacuolar system in health and disease call for the development of new tools allowing a better understanding of the complex molecular machinery involved in endocytic processes. We took advantage of the floating properties of small latex beads (sLB) on a discontinuous sucrose gradient to isolate highly purified endosomes following internalization of small latex beads in J774 macrophages and bone marrow-derived dendritic cells (DC). We particularly focused on the isolation of macrophages early endosomes and late endosomes/lysosomes (LE/LYS) as well as the isolation of LE/LYS from immature and lipopolysaccharide-activated (mature) DC. We subsequently performed a comparative analysis of their respective protein contents by MS. As expected, proteins already known to localize to the early endosomes were enriched in the earliest fraction of J774 endosomes, while proteins known to accumulate later in the process, such as hydrolases, were significantly enriched in the LE/LYS preparations. We next compared the LE/LYS protein contents of immature DC and mature DC, which are known to undergo massive reorganization leading to potent immune activation. The differences between the protein contents of endocytic organelles from macrophages and DC were underlined by focusing on previously poorly characterized biochemical pathways, which could have an unexpected but important role in the endosomal functions of these highly relevant immune cell types.     

Role of the low-selective organic anion transport in regulation of osmotic balance of renal collecting duct principal cells under hypo-osmotic conditions

In the course of adaptation of the rat kidney collecting duct cells to hypo-osmotic medium, the organic anion transporter inhibitor probenecid reduced significantly the regulatory cell volume decrease in response to a hypotonic shock. Both probenecid and hypotonic shock delayed significantly the entry into a cell of the fluorescent dye calcein, which exists as anion at neutral pH. Thus, the organic osmolyte transport plays an important role in the regulatory decrease of the principal cell volume under the hypo-osmotic conditions.     

Tubular lysosomes accompany stimulated pinocytosis in macrophages

A network of tubular lysosomes extends through the cytoplasm of J774.2 macrophages and phorbol ester-treated mouse peritoneal macrophages. The presence of this network is dependent upon the integrity of cytoplasmic microtubules and correlates with high cellular rates of accumulation of Lucifer Yellow (LY), a marker of fluid phase pinocytosis. We tested the hypothesis that the efficiency of LY transfer between the pinosomal and lysosomal compartments is increased in the presence of tubular lysosomes by asking how conditions that deplete the tubular lysosome network affect pinocytic accumulation of LY. Tubular lysosomes were disassembled in cells treated with microtubule-depolymerizing drugs or in cells that had phagocytosed latex beads. In unstimulated peritoneal macrophages, which normally contain few tubular lysosomes and which exhibit relatively inefficient transfer of pinocytosed LY to lysosomes, such treatments had little effect on pinocytosis. However, in J774 macrophages and phorbol ester-stimulated peritoneal macrophages, these treatments markedly reduced the efficiency of pinocytic accumulation of LY. We conclude that a basal level of solute accumulation via pinocytosis proceeds independently of the tubular lysosomes, and that an extended tubular lysosomal network contributes to the elevated rates of solute accumulation that accompany macrophage stimulation. Moreover, we suggest that the transformed mouse macrophage cell line J774 exhibits this stimulated pinocytosis constitutively.     

Secretory granules and endosomes show saltatory movement biased to the anterograde and retrograde directions, respectively, along microtubules in AtT20 cells

AtT20 (clone D16V) cells develop long neurite-like processes in the growth cones of which secretory granules containing ACTH accumulate. These secretory granules have an acidic pH. Using acridine orange as a vital stain for acidic organelles, in combination with video-enhanced fluorescence microscopy, and subsequent immunolabeling with rabbit antibodies against ACTH, we have shown that these secretory granules move by saltations along the processes. During saltations velocities of 3 to 5 microns/s are achieved. The majority of the secretory granules move in the anterograde direction but some move retrogradely. The growth cones and processes are the site of extensive endocytosis. Using Lucifer Yellow as a vital stain we have shown that most endosomes move by saltations retrogradely. Movement of both secretory granules and endosomes is dependent upon microtubules. Individual secretory granules or endosomes never reverse the direction of their movement as they traverse the processes. Neutralization of the lumen of these acidic organelles with NH4Cl does not inhibit their movement or change its direction.     

Inhibition of the acidification of endosomes and lysosomes by the antibiotic concanamycin B in macrophage J774.

The antibiotic concanamycin B was found to inhibit oxidized-low-density-lipoprotein(LDL)-induced accumulation of lipid droplets in the macrophage J774 at a concentration of 5-10 nM. Concanamycin B inhibited cholesteryl-ester synthesis from [14C]oleate by 50% at 14 nM without affecting the synthesis of triacylglycerol and polar lipids. Degradation of internalized oxidized 125I-LDL was inhibited by about 80% in cells treated with 25 nM concanamycin B, while cell-surface binding of oxidized 125I-LDL at 4 degrees C, uptake of surface-bound oxidized 125I-LDL and microsomal acyl-CoA:cholesterol acyltransferase activity were not significantly affected by the antibiotic at 25 nM. When J774 cells were treated with 25 nM concanamycin B at 37 degrees C for 60 min, there was a reduction of about 50% in the activity of cell-surface receptors. This reduction appeared to be due to partial trapping of the receptors within the cells. Concanamycin B significantly inhibited ATP-dependent acidification of endosomes and lysosomes of the J774 cells at a concentration of 4 nM. Since acidic condition of these organelles is required for receptor recycling and hydrolysis of lipoproteins, the results demonstrate that concanamycin-B inhibition of oxidized-LDL-induced accumulation of lipid droplets and cholesteryl esters in macrophages J774 is associated with reduced ATP-dependent acidification of these organelles.     

Effect of S-(2-chloroethyl)-DL-cysteine on the transport of p-aminohippurate ion in renal plasma membrane vesicles

The effects of S-(2-chloroethyl)-DL-cysteine (CEC) (a potent nephrotoxin) on the transport of p-aminohippurate ion (PAH) in renal plasma membrane vesicles isolated from rat renal cortex were studied in vitro. The uptake of PAH was significantly reduced in a dose-dependent manner in both the brush border membrane (BBM) and basolateral membrane (BLM) vesicles. These results demonstrate that CEC is capable of interfering with the accumulation of PAH (a model organic anion for renal tubular transport system) by both energy-independent and energy-dependent carrier-mediated transport processes. Probenecid, a typical inhibitor of the organic anion transport system, showed the highest inhibition of PAH uptake in both the membranes vesicles. These data indirectly suggest that transport by renal tubular cells may result in the accumulation of CEC in renal cellular organelles eventually in toxic concentrations. Thus, CEC showed both dose- and time-dependent inhibition of the activities of gamma-glutamyl transferase (a BBM marker enzyme) and Na+, K(+)-ATPase (a BLM marker enzyme), while no such inhibition was noticed with probenecid. Pretreatment with probenecid prevented the inhibition of the gamma-glutamyl transferase activity due to CEC in BBM, but failed to do so for the Na+,K(+)-ATPase activity in BLM vesicles. Thus, the data suggest that the inhibition of the activities of these membrane-specific enzymes by CEC could lead to the initial development of its nephrotoxicity.     

Products of endocytosis and autophagy are retrieved from axons by regulated retrograde organelle transport

Cellular homeostasis in neurons requires that the synthesis and anterograde axonal transport of protein and membrane be balanced by their degradation and retrograde transport. To address the nature and regulation of retrograde transport in cultured sympathetic neurons, I analyzed the behavior, composition, and ultrastructure of a class of large, phase-dense organelles whose movement has been shown to be influenced by axonal growth (Hollenbeck, P. J., and D. Bray. 1987. J. Cell Biol. 105:2827-2835). In actively elongating axons these organelles underwent both anterograde and retrograde movements, giving rise to inefficient net retrograde transport. This could be shifted to more efficient, higher volume retrograde transport by halting axonal outgrowth, or conversely shifted to less efficient retrograde transport with a larger anterograde component by increasing the intracellular cyclic AMP concentration. When neurons were loaded with Texas red- dextran by trituration, autophagy cleared the label from an even distribution throughout the neuronal cytosol to a punctate, presumably lysosomal, distribution in the cell body within 72 h. During this process, 100% of the phase-dense organelles were fluorescent, showing that they contained material sequestered from the cytosol and indicating that they conveyed this material to the cell body. When 29 examples of this class of organelle were identified by light microscopy and then relocated using correlative electron microscopy, they had a relatively constant ultrastructure consisting of a bilamellar or multilamellar boundary membrane and cytoplasmic contents, characteristic of autophagic vacuoles. When neurons took up Lucifer yellow, FITC-dextran, or Texas red-ovalbumin from the medium via endocytosis at the growth cone, 100% of the phase-dense organelles became fluorescent, demonstrating that they also contain products of endocytosis. Furthermore, pulse-chase experiments with fluorescent endocytic tracers confirmed that these organelles are formed in the most distal region of the axon and undergo net retrograde transport. Quantitative ratiometric imaging with endocytosed 8-hydroxypyrene-1,3,6- trisulfonic acid showed that the mean pH of their lumena was 7.05. These results indicate that the endocytic and autophagic pathways merge in the distal axon, resulting in a class of predegradative organelles that undergo regulated transport back to the cell body.     

Degradation of epidermal growth factor receptor in rat liver. Membrane topology through the lysosomal pathway.

We have generated and characterized three rabbit polyclonal antibodies that recognize different regions of the epidermal growth factor receptor (EGF-R) and used them to study the degradation of the receptor in the isolated perfused rat liver. Quantitative immunoblot analyses of rat liver homogenates prepared from tissue biopsies collected at various times after epidermal growth factor (EGF) addition showed that both the ectoplasmic and cytoplasmic domains of rat liver EGF-Rs were degraded with similar kinetics (t1/2 = 3.5-3.8 h at 25 degrees C with cycloheximide). No immunoreactive intermediate breakdown products were detected. EGF-stimulated degradation of both receptor domains was inhibited by the thiol protease inhibitor leupeptin, suggesting lysosome involvement in the hydrolysis of the whole molecule. To study this further, protease protection experiments were performed on endosome- and lysosome-enriched fractions isolated from leupeptin-treated livers. We found that the cytoplasmic domains of greater than 90% of the EGF-Rs in endosomal fractions were accessible to digestion when proteinase K was added to the intact vesicle populations, while the ectoplasmic domain was unaltered. In contrast, both the ectoplasmic and cytoplasmic domains of approximately 55% of the EGF-Rs present in lysosome-enriched fractions were inaccessible to proteinase K digestion in the absence of detergent. These findings suggest that movement of EGF-Rs from the limiting membrane of endosomes to the lumen of lysosomes permits the degradation of the entire EGF-R molecule within lysosomes.     

Chasing the elusive mammalian microautophagy

Different mechanisms for delivery of intracellular components (proteins and organelles) to lysosomes and late endosomes for degradation co-exist in almost all cells and set the basis for distinct autophagic pathways. Cargo can be sequestered inside double-membrane vesicles (or autophagosomes) and reach the lysosomal compartment upon fusion of these vesicles to lysosomes through macroautophagy. In a different type of autophagy, known as chaperone-mediated autophagy (CMA), single individual soluble proteins can be targeted one by one to the lysosomal membrane and translocated into the lumen for degradation. Direct sequestration of proteins and organelles by invaginations at the lysosomal membrane that pinch off into the lumen has also been proposed. This process, known as microautophagy, remains poorly understood in mammalian cells. In our recent work, we demonstrate the occurrence of both "in bulk" and "selective" internalization of cytosolic components in late endosomes and identify some of the molecular players of this process that we have named endosomalmicroautophagy (e-MI) due to its resemblance to microautophagy.     

In vivo fluorescence imaging of the functional organization of trophotaenial placental cells of goodeid fishes

Embryos of viviparous goodeid fishes undergo a 10 to 150 × increase in dry weight during gestation. Maternal nutrients are transferred across a trophotaenial placenta comprised of the ovarian lumenal epithelium and the trophotaeniae of the embryo. Trophotaeniae are externalized projections of the embryonic hindgut. Epithelial cells of the ribbon trophotaenia (Ameca splendens) resemble intestinal absorptive cells of suckling mammals and endocytose macromolecules. They possess an apical brush border, endocytotic complex, endosomal–lysosomal system, and apical and basal clusters of mitochondria. Cells of the rosette trophotaenia (Goodea atripinnis) lack an endocytotic apparatus, have small lysosomes, two mitochondrial clusters, and transport small molecules. Organelle-specific fluorescent probes were employed to characterize the functional organization of the two types of trophotaenial cells. In A. splendens, Lucifer Yellow, a membrane-impermeable tracer of vesicular transport, first appears in peripheral vesicles (15–45 sec), then passes into elongated tubular endosomes (1–3 min) and later appears in large central vacuoles (10–15 min). These vacuoles accumulate Acridine Orange, a classical probe for lysosomes, and have been shown to contain lysosomal enzymes. Endosomelysosome fusion was observed. In both A. splendens and G. atripinnis, Rhodamine 123 fluorescence was localized in two clusters of fine spots that corresponded to mitochondria. 4′,6-diaminido-2-phenyl-indole (DAPI) staining of nuclei established the positional relationships of cell organelles with respect to the nuclei. 3,3′-dihexyloxacarbo-cyanine iodide (DiOC₆) revealed the perinuclear distribution of the endoplasmic reticulum. In order to compare in vivo fluorescence of Lucifer Yellow with previous ultrastructural observations, we employed fluorescence photoconversion and electron microscopy. © 1994 Wiley-Liss, Inc.     

Overexpression of the Dynamitin (p50) Subunit of the Dynactin Complex Disrupts Dynein-dependent Maintenance of Membrane Organelle Distribution

Dynactin is a multisubunit complex that plays an accessory role in cytoplasmic dynein function. Overexpression in mammalian cells of one dynactin subunit, dynamitin, disrupts the complex, resulting in dissociation of cytoplasmic dynein from prometaphase kinetochores, with consequent perturbation of mitosis (Echeverri, C.J., B.M. Paschal, K.T. Vaughan, and R.B. Vallee. 1996. J. Cell Biol. 132:617–634). Based on these results, dynactin was proposed to play a role in linking cytoplasmic dynein to kinetochores and, potentially, to membrane organelles. The current study reports on the dynamitin interphase phenotype. In dynamitin-overexpressing cells, early endosomes (labeled with antitransferrin receptor), as well as late endosomes and lysosomes (labeled with anti–lysosome-associated membrane protein-1 [LAMP-1]), were redistributed to the cell periphery. This redistribution was disrupted by nocodazole, implicating an underlying plus end–directed microtubule motor activity. The Golgi stack, monitored using sialyltransferase, galactosyltransferase, and N-acetylglucosaminyltransferase I, was dramatically disrupted into scattered structures that colocalized with components of the intermediate compartment (ERGIC-53 and ERD-2). The disrupted Golgi elements were revealed by EM to represent short stacks similar to those formed by microtubule-depolymerizing agents. Golgi-to-ER traffic of stack markers induced by brefeldin A was not inhibited by dynamitin overexpression. Time-lapse observations of dynamitin-overexpressing cells recovering from brefeldin A treatment revealed that the scattered Golgi elements do not undergo microtubule-based transport as seen in control cells, but rather, remain stationary at or near their ER exit sites. These results indicate that dynactin is specifically required for ongoing centripetal movement of endocytic organelles and components of the intermediate compartment. Results similar to those of dynamitin overexpression were obtained by microinjection with antidynein intermediate chain antibody, consistent with a role for dynactin in mediating interactions of cytoplasmic dynein with specific membrane organelles. These results suggest that dynamitin plays a pivotal role in regulating organelle movement at the level of motor–cargo binding.