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.     

pH-induced microtubule-dependent redistribution of late endosomes in neuronal and epithelial cells

The interaction between late endocytic structures and microtubules in polarized cells was studied using a procedure previously shown to cause microtubule-dependent redistribution of lysosomes in fibroblasts and macrophages (Heuser, J. 1989. J. Cell Biol. 108:855-864). In cultured rat hippocampal neurons, low cytoplasmic pH caused cation-independent mannose-6-phosphate receptor-enriched structures to move out of the cell body and into the processes. In filter grown MDCK cells lowering the cytosolic pH to approximately 6.5 caused late endosomes to move to the base of the cell and this process was shown to be microtubule dependent. Alkalinization caused a shift in distribution towards the apical pole of the cell. The results are consistent with low pH causing the redistribution of late endosomes towards the plus ends of the microtubules. In MDCK cells the microtubules orientated vertically in the cell may play a role in this process. The shape changes that accompanied the redistribution of the late endosomes in MDCK cells were examined by electron microscopy. On low pH treatment fragmentation of the late endosomes was observed whereas after microtubule depolymerization individual late endosomal structures appeared to fuse together. The late endosomes of the MDCK cell appear to be highly pleomorphic and dependent on microtubules for their form and distribution in the cell.     

Regulation of kinesin-directed movements

Bidirectional organelle transport along microtubules is most likely mediated by the opposing forces generated by two microtubule-based motors: kinesin and cytoplasmic dynein. Because the direction and timing of organelle movements are controlled by the cell, the activity of one or both of these motor molecules must be regulated. Recent studies demonstrate that kinesin, kinesin-like proteins and kinesin-associated proteins can be phosphorylated, and suggest that changes in their phosphorylation state may modulate kinesin's ability to interact with either microtubules or organelles. Thus, it is possible that phosphorylation regulates kinesin-driven movements.     

Cell cycle control of microtubule-based membrane transport and tubule formation in vitro

When higher eukaryotic cells enter mitosis, membrane organization changes dramatically and traffic between membrane compartments is inhibited. Since membrane transport along microtubules is involved in secretion, endocytosis, and the positioning of organelles during interphase, we have explored whether the mitotic reorganization of membrane could involve a change in microtubule-based membrane transport. This question was examined by reconstituting organelle transport along microtubules in Xenopus egg extracts, which can be converted between interphase and metaphase states in vitro in the absence of protein synthesis. Interphase extracts support the microtubule-dependent formation of abundant polygonal networks of membrane tubules and the transport of small vesicles. In metaphase extracts, however, the plus end- and minus end-directed movements of vesicles along microtubules as well as the formation of tubular membrane networks are all reduced substantially. By fractionating the extracts into soluble and membrane components, we have shown that the cell cycle state of the supernatant determines the extent of microtubule-based membrane movement. Interphase but not metaphase Xenopus soluble factors also stimulate movement of membranes from a rat liver Golgi fraction. In contrast to above findings with organelle transport, the minus end-directed movements of microtubules on glass surfaces and of latex beads along microtubules are similar in interphase and metaphase extracts, suggesting that cytoplasmic dynein, the predominant soluble motor in frog extracts, retains its force-generating activity throughout the cell cycle. A change in the association of motors with membranes may therefore explain the differing levels of organelle transport activity in interphase and mitotic extracts. We propose that the regulation of organelle transport may contribute significantly to the changes in membrane structure and function observed during mitosis in living cells.     

Actin Filaments and Myosin I Alpha Cooperate with Microtubules for the Movement of Lysosomes

An earlier report suggested that actin and myosin I alpha (MMIalpha), a myosin associated with endosomes and lysosomes, were involved in the delivery of internalized molecules to lysosomes. To determine whether actin and MMIalpha were involved in the movement of lysosomes, we analyzed by time-lapse video microscopy the dynamic of lysosomes in living mouse hepatoma cells (BWTG3 cells), producing green fluorescent protein actin or a nonfunctional domain of MMIalpha. In GFP-actin cells, lysosomes displayed a combination of rapid long-range directional movements dependent on microtubules, short random movements, and pauses, sometimes on actin filaments. We showed that the inhibition of the dynamics of actin filaments by cytochalasin D increased pauses of lysosomes on actin structures, while depolymerization of actin filaments using latrunculin A increased the mobility of lysosomes but impaired the directionality of their long-range movements. The production of a nonfunctional domain of MMIalpha impaired the intracellular distribution of lysosomes and the directionality of their long-range movements. Altogether, our observations indicate for the first time that both actin filaments and MMIalpha contribute to the movement of lysosomes in cooperation with microtubules and their associated molecular motors.     

The C-terminus of tubulin increases cytoplasmic dynein and kinesin processivity

In motor movement on microtubules, the anionic C-terminal of tubulin has been implicated as a significant factor. Our digital analyses of movements of cytoplasmic dynein- and kinesin-coated beads on microtubules have revealed dramatic changes when the C-terminal region (2-4-kDa fragment) of tubulin was cleaved by limited subtilisin digestion of assembled microtubules. For both motors, bead binding to microtubules was decreased threefold, bead run length was decreased over fourfold, and there was a dramatic 20-fold decrease in diffusional movements of cytoplasmic dynein beads on microtubules (even with low motor concentrations where the level of bead motile activity was linear with motor concentration). The velocity of active bead movements on microtubules was unchanged for cytoplasmic dynein and slightly decreased for kinesin. There was also a decrease in the frequency of bead movements without a change in velocity when the ionic strength was raised. However, with high ionic strength there was not a decrease in run length or any selective inhibition of the diffusional movement. The C-terminal region of tubulin increased motor run length (processivity) by inhibiting "detachment" but without affecting velocity. Because the major motor binding sites of microtubules are not on the C-terminal tail of tubulin (), we suggest that the changes are the result of the compromise of a weakly attached state that is the lowest affinity step in both motors' ATPase cycles and is not rate limiting.     

受体介导内吞引起的巨噬细胞胞质酸化和胞吐作用

本文利用激光扫描共聚焦显微镜A-CAS570从细胞形态学和功能两方面,研究了刀豆素A(Concanavalin A,Con A)、麦芽凝集素(Wheat Germ Agglutinin,WGA)、酵母多糖(Zymosan A,Z.A)对小鼠腹腔巨噬细胞胞质pH和溶酶体内荧光探针FITC—Dextran排出细胞的影响。结果显示三种配体加入细胞外液10min内,胞质pH很快下降,此后维持在该水平;在15min左右细胞外FITC一Dextran迅速增加,20min后变化趋于停止;在三种配体加入后15min左右,细胞内溶酶体在质膜内侧增多;25—30min溶酶体重新向细胞中央运动。根据上述实验结果,我们认为溶酶体pH升高是触发溶酶体内荧光探针通过胞吐作用排出细胞的必要条件,胞质酸化抑制溶酶体内容物通过胞吐作用排出细胞。配体刺激引起的溶酶体内容物通过胞吐作用排出细胞和胞质酸化是细胞自我调节和保护的一种反映。     

受体介导内吞引起的巨噬细胞胞质酸化和胞吐作用

The effects of Con A, WGA, Zymosan A on macrophage cytosolic pH and outflow of lysosomal content through exocytosis were studied with SNAFL-calcein and FITC-Dextran on ACAS570. The results showed all three ligands could induce macrophage cytosolic acidification in about 10 min and kept at the same level hereafter; outflow of lysosomal fluorescent probe through exocytosis appeared in 15-20 min. In resting conditions, macrophage lysosomes mainly distributed in cell center; after stimulated for 15 min by three ligands, the number of lysosomes increased in membrane periphery, in 25-30 min lysosomes moved back toward cell center. We proposed that ligands induced lysosomal pH rises was a basic factor for outflow of lysosomal content through exocytosis, cytosolic acidification inhibited receptor-mediated endocytosis. Cytosolic acidification and outflow of lysosomal content through exocytosis were the results of cellular self-regulation and self-protection during receptor-mediated endocytosis.     

The morphology but not the function of endosomes and lysosomes is altered by brefeldin A

Brefeldin A (BFA) induces the formation of an extensively fused network of membranes derived from the trans-Golgi network (TGN) and early endosomes (EE). We describe in detail here the unaffected passage of endocytosed material through the fused TGN/EE compartments to lysosomes in BFA-treated cells. We also confirmed that BFA caused the formation of tubular lysosomes, although the kinetics and extent of tubulation varied greatly between different cell types. The BFA-induced tubular lysosomes were often seen to form simple networks. Formation of tubular lysosomes was microtubule-mediated and energy-dependent; interestingly, however, maintenance of the tubulated lysosomes only required microtubules and was insensitive to energy poisons. Upon removal of BFA, the tubular lysosomes rapidly recovered in an energy-dependent process. In most cell types examined, the extensive TGN/EE network is ephemeral, eventually collapsing into a compact cluster of tubulo-vesicular membranes in a process that precedes the formation of tubular lysosomes. However, in primary bovine testicular cells, the BFA-induced TGN/EE network was remarkably stable (for > 12 h). During this time, the TGN/EE network coexisted with tubular lysosomes, however, the two compartments remained completely separate. These results show that BFA has multiple, profound effects on the morphology of various compartments of the endosome-lysosome system. In spite of these changes, endocytic traffic can continue through the altered compartments suggesting that transport occurs through noncoated vesicles or through vesicles that are insensitive to BFA.     

THE ASSOCIATION OF A CLASS OF SALTATORY MOVEMENTS WITH MICROTUBULES IN CULTURED CELLS

Particulate structures in the cytoplasm of HeLa and other cultured cells in interphase undergo rapid individual linear displacements (long saltatory movements, LSM). By the use of time-lapse microscopy to locate saltating particles prior to fixation and histochemical examination of the cells, structures of several kinds have been shown to move in this manner. Elements that show LSM include lysosomes, pinosomes, ingested carbon particles, lipoidal granules, and unidentified particles that appear as bright objects in positive phase contrast. The pattern of movement of the particles suggests the presence of linear guiding elements radially disposed from the cytocenter (centriole region). The participation of microtubules in these movements is inferred from the observation that LSM cease after treatment with drugs which depolymerize microtubules, i.e., colchicine, Vinblastine, and podophyllin. The directions of the microtubules in the cytoplasm of HeLa cells found by electron microscopy are consistent with the aster-like configuration predicted from study of LSM. Further support for this arrangement of cytoplasmic microtubules is provided by light microscope observations of colchicine-sensitive radial arrays of acid phosphatase granules in the cytoplasm of some cell lines.     

Lysosome dispersion in osteoblasts accommodates enhanced collagen production during differentiation

Lysosomes are essential organelles for intracellular degradation and are generally sequestered near the cell center to receive vesicles with contents targeted for destruction. During ascorbic acid (AA)-induced differentiation of osteogenic cells ( Beck, G. R., Jr., Zerler, B., and Moran, E. (2001) Cell Growth Differ. 12, 61-83 ), we saw a marked increase in total lysosome organelles in osteoblastic cells, in addition to an enhanced endocytic rate. Interestingly, lysosomes were dispersed toward the cell periphery in differentiating osteoblasts. We determined that lysosome dispersion in differentiated osteoblasts required intact microtubules for long range transport and was dependent on kinesin motors but did not involve cytosolic acidification. Impairment of lysosome dispersion markedly reduced AA-induced osteoblast differentiation. Lysosomes were not secreted in differentiated osteoblasts, implicating them instead in intracellular degradation. We assayed the degradative capacity and saw a significant increase in DQ-ovalbumin fluorescence in differentiated osteogenic cells compared with undifferentiated control cells. Osteogenic cells are specialized for type I collagen production, and we noted enhanced secreted and intracellular collagen in AA-differentiated osteoblasts versus control cells. Importantly, osteoblasts displayed procollagen-containing vesicles that were distributed throughout the cytoplasm, a portion of which colocalized with lysosomes. Treatment of cells with 2,2'-dipyridyl to inhibit procollagen trimerization enhanced colocalization of lysosomes with procollagen-containing organelles, implicating dispersed lysosomes in collagen processing in osteogenic cells.     

Secondary lysosomes as an integral part of the cytoskeleton: a morphological study in rat Kupffer cells

Rat Kupffer cells contain the three major cytoskeletal components: microfilaments (MF), microtubules (MT), and intermediate filaments (IF) of the vimentin type. Previous cytomagnetometric data obtained from alveolar macrophages and rat Kupffer cells in culture provided evidence that actin filaments contribute to the movements of lysosomes. The lysosomal transport in living cells was affected, when the MFs were selectively disturbed, whereas the depolymerization of the MTs had no effect on the lysosomal movement measured by cytomagnetometric means. Immunofluorescence and ultrastructural studies of isolated and cultured rat Kupffer cells, presented in this paper, will investigate the relationship between lysosomes and the cytoskeleton. The principal filamentous structure in the peripheral cytoplasm of Kupffer cells in a dense meshwork of actin filaments. The dimension of the meshes combined with the dimensions of lysosomes implies the necessity of either (i) disintegration of the actin filament cross-links, (ii) depolarymerization and redistribution of MF's, or (iii) a displacement of actin filaments by the lysosomes during the organelle transport. The presence of microtubules in cytoplasmic protrusions and their track from the periphery to the perinuclear region during interphase might play a role in the transport mechanism of lysosomes, the more so because microtubules could often be demonstrated in closest association with lysosomes even in the first phase of endocytosis. The distribution pattern of vimentin, found as a dense interconnected framework surrounding the lysosomes like a basket, could play a role in positioning the organelles. The dynamic functions of MF's and MT's and their multifunctionality led to an adaptive and flexible organization of these filaments which may both be involved in lysosomal motion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vav1 and Vav2 play different roles in macrophage migration and cytoskeletal organization

Vav family proteins act as guanine nucleotide exchange factors for Rho family proteins, which are known to orchestrate cytoskeletal changes and cell migration in response to extracellular stimuli. Using mice deficient for Vav1, Vav2 and/or Vav3, overlapping and isoform-specific functions of the three Vav proteins have been described in various hematopoietic cell types, but their roles in regulating cell morphology and migration have not been studied in detail. To investigate whether Vav isoforms have redundant or unique functions in regulating adhesion and migration, we investigated the properties of Vav1-deficient and Vav2-deficient macrophages. Both Vav1-deficient and Vav2-deficient cells have a smaller adhesive area; yet, only Vav1-deficient cells have a reduced migration speed, which coincides with a lower level of microtubules. Vav2-deficient macrophages display a high level of constitutive membrane ruffling, but neither Vav1 nor Vav2 is required for colony stimulating factor-1-induced membrane ruffling and cell spreading. Our results suggest that the migration speed of macrophages is regulated independently of spread area or membrane ruffling and that Vav1 is selectively required to maintain a normal migration speed.     

Tubulin-specific antibody and the expression of microtubules in 3T3 cells after attachment to a substratum : Further evidence for the polar growth of cytoplasmic microtubules in vivo

Mouse 3T3 cells were allowed to attach to and spread on glass. The expression of cytoplasmic microtubules during the respreading process was monitored by immunofluorescence microscopy using monospecific antibody against tubulin. During radial attachment of the cells a ring of flattened cytoplasm is seen around the nucleus. Cytoplasmic microtubules then enter this spreading ring from the perinuclear region and elongate toward the plasma membrane. At later times microtubules appear perpendicular to the plasma membrane and seem to be in intimate contact with it giving the impression that they “stretch” the cytoplasm. When the cells assume their typical fibroblastic shape numerous microtubules are seen. They traverse the cytoplasm. Some come close to the plasma membrane and some bend to conform to the shape of the cell. Changes in microtubular organization correlate well with changes in cell shape. These results together with our previous observations on the assembly of cytoplasmic microtubules upon recovery from colcemid treatment suggest that microtubules may grow as polar structures from a microtubular organizing center towards the plasma membrane. The hypothesis that cytoplasmic microtubules might confer polarity on the cell is discussed.     

Motor and cargo interactions.

The movements of intracellular cargo along microtubules within cells are often saltatory or of short duration. Further, calculations of the fraction of membrane vesicles that are moving at any period, indicate that active motor complexes are rare. From observations of normal vesicle traffic in cells, there appears to be position-dependent activation of motors and a balance of traffic in the inward and outward directions. In-vitro binding of motors to cargo is observed under many conditions but motility is not. Multi-component complexes appear to be involved in producing active organelle movements by a graded activation system that is highly localized in the cell. The basis of the activation of motility of the organelle motor complexes is still unknown but phosphorylation has been implicated in many systems. In the case of the motor-binding protein, kinectin, it has been linked to active organelle movements powered by conventional kinesin. From the coiled-coil structure of kinectin and the coiled-coil tail of kinesin, it is postulated that a coiled-coil assembly is responsible for the binding interaction. Many other cargoes are transported but the control of transport will be customized for each function, such as axonemal rafts or cytoskeletal complexes. Each function will have to be analyzed separately and motor activity will need to be integrated into the specific aspects of the function.     

Heterotrimeric Kinesin II Is the Microtubule Motor Protein Responsible for Pigment Dispersion in Xenopus Melanophores

Melanophores move pigment organelles (melanosomes) from the cell center to the periphery and vice-versa. These bidirectional movements require cytoplasmic microtubules and microfilaments and depend on the function of microtubule motors and a myosin. Earlier we found that melanosomes purified from Xenopus melanophores contain the plus end microtubule motor kinesin II, indicating that it may be involved in dispersion (Rogers, S.L., I.S. Tint, P.C. Fanapour, and V.I. Gelfand. 1997. Proc. Natl. Acad. Sci. USA. 94: 3720–3725). Here, we generated a dominant-negative construct encoding green fluorescent protein fused to the stalk-tail region of Xenopus kinesin-like protein 3 (Xklp3), the 95-kD motor subunit of Xenopus kinesin II, and introduced it into melanophores. Overexpression of the fusion protein inhibited pigment dispersion but had no effect on aggregation. To control for the specificity of this effect, we studied the kinesin-dependent movement of lysosomes. Neither dispersion of lysosomes in acidic conditions nor their clustering under alkaline conditions was affected by the mutant Xklp3. Furthermore, microinjection of melanophores with SUK4, a function-blocking kinesin antibody, inhibited dispersion of lysosomes but had no effect on melanosome transport. We conclude that melanosome dispersion is powered by kinesin II and not by conventional kinesin. This paper demonstrates that kinesin II moves membrane-bound organelles.     

Phorbol myristate acetate stimulates microtubule and 10-nm filament extension and lysosome redistribution in mouse macrophages

Phorbol myristate acetate (PMA) stimulates cell spreading and fluid- phase pinocytosis in mouse peritoneal macrophages. Colchicine (10(-5) M) and cytochalasin B (10(-5) M) abolish PMA stimulated pinocytosis but have little effect on cellular spreading (Phaire-Washington et al., 1980, J. Cell Biol., 86:634-640). We report here that PMA also alters the organization of the cytoskeleton and the distrubution of organelles in these cells. Neither control nor PMA-treated macrophages contain actin cables. PMA-treated resident thioglycolate-elicited macrophages exhibit beneath their substrate-adherent membranes many randomly distributed punctate foci that stain brightly for actin. The appearance and distribution of these actin-containing foci are not altered by colchicine (10(-5) M) or cytochalasin B (10(-5) M). In thioglycolate- elicited macrophages PMA causes the extension and radial organization of microtubules and 10-nm filaments and promotes the movement of secondary lysosomes from their perinuclear location to the peripheral cytoplasm. Depending upon the concentration of PMA used, 45-71% of thioglycolate-elicited macrophages and 32-44% of proteose-peptone- elicited macrophages and numerous lysosomes, radiating from the centrosphere region, arranged linearly along microtubule and 10-nm filament bundles. Colchicine (10(-5) M) and podophyllotoxin (10(-5) M) prevent the radial redistribution of microtubules, 10-nm filaments, and lysosomes in these cells. Cytochalasins B and D (10(-5) M) have no inhibitory effects on these processes. These findings indicate that microtubules and 10-nm filaments respond in a coordinated fashion to PMA and to agents that inhibit microtubule function; they suggest that these cytoskeletal elements regulate the movement and distribution of lysosomes in the macrophage cytoplasm.     

Actin cytoskeleton controls movement of intracellular organelles in pancreatic duct epithelial cells

In most eukaryotic cells, microtubules and filamentous actin (F-actin) provide tracks on which intracellular organelles move using molecular motors. Here we report that cytoplasmic movement of both mitochondria and lysosomes is slowed by F-actin meshwork formation in pancreatic duct epithelial cells (PDEC). Mitochondria and lysosomes were labeled with fluorescent Mitotracker Red CMXRos and Lysotracker Red DND-99, respectively, and their movements were monitored using epi-fluorescence and confocal microscopy. Mitochondria and lysosomes moving actively at rest stopped rapidly within several seconds after an intracellular Ca(2+) rise induced by activation of P2Y(2) purinergic receptors. The 'freezing' of the organelles was inhibited by blocking the Ca(2+) rise or by pretreatment with latrunculin B, an inhibitor of F-actin formation. Indeed, this freezing effect on the organelles was accompanied by the formation of F-actin in the whole cytoplasm as stained with Alexa 488-phalloidin in fixed PDEC. For real-time monitoring of F-actin formation in live cells, we expressed sGFP-fimbrin actin binding domain2 (fABD2) in PDEC. Rapid recruitment of the fluorescent probe near the nucleus and lysosomes suggested dense F-actin formation around intracellular structures. The development of F-actin paralleled that of organelle freezing. We conclude that rapid Ca(2+)-dependent F-actin formation physically restrains intracellular organelles and reduces their mobility non-selectively in PDEC.     

Ultrastructure of intermediate stages in polarity reversal of thyroid epithelium in follicles in suspension culture

Separated thyroid follicles can be maintained in suspension culture in Coon's modified F-12 medium in 0.5% calf serum. If the serum concentration is raised to 5%, the follicles undergo inversion in 3-5 d. During the process of inversion, epithelial cells can be observed in intermediate stages of polarity reversal. The earliest ultrastructural changes recognized are surface changes in which tight junctions and microvilli appear at the lateral margins of the cell near the medium. Later, changes in the distribution of intracellular organelles occur. The Golgi apparatus shifts towards the end of the cell facing the medium, and lysosomes shift toward the luminal end of the cell. The right junctions and microvilli at the luminal end of the cell disappear sometime after the cytoplasmic organelles rearrange. The luminal colloid disappears only after the surface changes (loss of tight junctions and microvilli) occur at the luminal end of the cell. There appears to be some regulation of the order in which changes occur during polarity reversal of the thyroid epithelial cell.     

Na+/H+ Exchangers and RhoA Regulate Acidic Extracellular pH-Induced Lysosome Trafficking in Prostate Cancer Cells

Acidic extracellular pH (pHe) is a common feature of the tumor microenvironment and has been implicated in tumor invasion through the induction of protease secretion. Since lysosomes constitute the major storehouse of cellular proteases, the trafficking of lysosomes to the cell periphery may be required in order to secrete proteases. We demonstrate that a pHe of 6.4-6.8 induced the trafficking of lysosomes to membrane protrusions in the cell periphery. This trafficking event depended upon the PI3K pathway, the GTPase RhoA and sodium-proton exchange activity, resulting in lysosomal exocytosis. Acidic pHe induced a cytoplasmic acidification (although cytoplasmic acidification was not sufficient for acidic pHe-induced lysosome trafficking and exocytosis) and inhibition of NHE activity with the amiloride derivative, EIPA or the anti-diabetic agent troglitazone prevented lysosome trafficking to the cell periphery. Interestingly, using the more specific NHE1 and NHE3 inhibitors, cariporide and s3226 respectively, we show that multiple NHE isoforms are involved in acidic pHe-induced lysosome trafficking and exocytosis. Moreover, in cells expressing NHE1 shRNA, although basal NHE activity was decreased, lysosomes still underwent acidic pHe-induced trafficking, suggesting compensation by other NHE family members. Together these data implicate proton exchangers, especially NHE1 and NHE3, in acidic pHe-induced lysosome trafficking and exocytosis.