Osmotically induced cell volume changes alter anterograde and retrograde transport, Golgi structure, and COPI dissociation

Abstract

Physiological conditions that impinge on constitutive traffic and affect organelle structure are not known. We report that osmotically induced cell volume changes, which are known to occur under a variety of conditions, rapidly inhibited endoplasmic reticulum (ER)-to-Golgi transport in mammalian cells. Both ER export and ER Golgi intermediate compartment (ERGIC)-to-Golgi trafficking steps were blocked, but retrograde transport was active, and it mediated ERGIC and Golgi collapse into the ER. Extensive tubulation and relatively rapid Golgi resident redistribution were observed under hypo-osmotic conditions, whereas a slower redistribution of the same markers, without apparent tubulation, was observed under hyperosmotic conditions. The osmotic stress response correlated with the perturbation of COPI function, because both hypo- and hyperosmotic conditions slowed brefeldin A-induced dissociation of betaCOP from Golgi membranes. Remarkably, Golgi residents reemerged after several hours of sustained incubation in hypotonic or hypertonic medium. Reemergence was independent of new protein synthesis but required PKC, an activity known to mediate cell volume recovery. Taken together these results indicate the existence of a coupling between cell volume and constitutive traffic that impacts organelle structure through independent effects on anterograde and retrograde flow and that involves, in part, modulation of COPI function.

Figure 1

ER-to-Golgi transport is sensitive to changes in extracellular osmolarity. (A) Both hypo- and hyperosmotic conditions block ER-to-Golgi transport. COS-7 cells transfected with HA epitope-tagged DPPIV were metabolically labeled for 30 min, chased in normal medium for 10 min, and placed in normal medium, hypotonic medium, hypertonic medium, or normal medium plus 10 μg/ml BFA for 110 min at 37°C. The DPPIV from each plate was isolated and analyzed for the acquisition of endo H resistance as described in MATERIALS AND METHODS. Endo H-sensitive (s) and resistant (r) forms of DPPIV are marked. (B) The response of ER-to-Golgi transport to extracellular osmolarity is threshold in nature. The assay in A was performed under a range of osmotic (media compositions as detailed in MATERIALS AND METHODS) conditions.

Figure 2

Hypo-osmotic conditions block the export of VSV-G from the ER. CHO cells were infected with ts-O45-VSV as described in MATERIALS AND METHODS and maintained at 39.5°C for 4 h to trap VSV-G in the ER. During the last 30 min of the 39.5°C incubation and in all subsequent incubations, 100 μg/ml cycloheximide was added to prevent new protein synthesis. Cells were shifted to 32°C in normal medium (B) or hypotonic (210-mOsm)medium (C) or maintained at 39.5°C (A) for 15 min, after which cells were fixed and processed for immunofluorescence using a mAb against VSV-G.

Figure 3

Hypo-osmotic conditions block transport of VSV-G from the ERGIC to the Golgi and causes VSV-G to redistribute from the ERGIC to the ER. CHO cells were infected with ts-O45-VSV and maintained at 39.5°C for 4 h. During the last 30 min of the 39.5°C incubation and in all subsequent incubations, 100 μg/ml cycloheximide was added to prevent new protein synthesis. Cells were then shifted to 15°C in normal medium to allow VSV-G to move into but not out of the ERGIC. After 1.5 h (see A), cells were shifted to 32°C in normal medium (B and C) or hypotonic (210-mOsm) medium (E and F) for 20 min (B and E) or 90 min (C and F) or maintained in normal medium at 15°C for 90 min (D). Cells were fixed and processed for immunofluorescence using a mAb against VSV-G.

Figure 4

Hypo-osmotic conditions induce the redistribution of ERGIC 53 to the ER. HeLa cells were grown to 50% confluence on 12-mm glass coverslips. Coverslips were removed from normal medium and placed in 1 ml of hypotonic (210-mOsm) medium for 15 min (B) or 30 min (C). Cells maintained in normal medium are shown in A, and cells incubated in normal medium with 10 μg/ml BFA for 60 min are shown in D. Cells were fixed and processed for immunofluorescence using a mAb against ERGIC 53.

Figure 5

Hypo-osmotic conditions induce the tubulation and redistribution of GPP130 to the ER. COS-7 cells, grown to 50% confluence on 12-mm glass coverslips, were removed from normal medium and placed in 1 ml of hypotonic (210-mOsm) medium for 30 min (B) or 120 min (C) or hypotonic medium plus 0.1 M sucrose (D) for 30 min. Cells maintained in normal medium are shown in A. Cells were fixed and processed for immunofluorescence using a mAb against GPP130.

Figure 6

Hypo-osmotic conditions induce the redistribution of other Golgi residents to the ER. HeLa cells, grown to 50% confluence on 12-mm glass coverslips, were removed from normal medium and placed in 1 ml of hypotonic medium for 5 min (B and F), 30 min (C and G), or 120 min (D and H). The inset in B shows an enlargement of a portion of the cell shown in B. Cells maintained in normal medium are shown in A and E. Cells were fixed and processed for double-label immunofluorescence using a polyclonal antiserum against GM130 (A–D) and a mAb against GPP130 (E–H).

Figure 7

Density gradient fractionation of ER and Golgi in untreated (A), BFA-treated (B), and hypotonically treated (C) cells. Fifteen-centimeter cm plates of confluent HeLa cells were incubated in normal medium without serum (A), normal medium without serum in the presence of 10 μg/ml BFA (B), or hypotonic medium (210 mOsm) (C) for 60 min at 37°C. The postnuclear supernatant obtained from each plate was fractionated in a sucrose step flotation gradient (as described in MATERIALS AND METHODS) and probed with antibodies against GPP130 (Golgi) and p63 (ER).

Figure 8

Hypotonic treatment prevents the rapid, BFA-induced dissociation of βCOP from the Golgi. HeLa cells, grown on 12-mm glass coverslips, were left in normal medium (A, B, D, and E) or placed in 1 ml of hypotonic medium for 1 min (C and F) before the addition of 10 μg/ml BFA. Two minutes after BFA addition, cells were fixed and processed for double-label immunofluorescence using a mAb against βCOP (A–C) and a polyclonal antiserum against giantin (D–F).

Figure 9

Effect of hypotonic and hypertonic treatment on the rate of βCOP dissociation (A) and association (B). (A) HeLa cells, grown on 12-mm glass coverslips, were left in normal medium without serum or placed in hypotonic medium (210 mOsm) or hypertonic medium (610 mOsm) for 1 min before the addition of 10 μg/ml BFA. At each time point indicated, cells were fixed and stained using a mAb against βCOP and a polyclonal antiserum against giantin. The average of the mean fluorescence intensity of βCOP staining in a fixed circle (2782 pixels) enclosing the Golgi region (as indicated by giantin staining) in 50 cells was measured. (B) HeLa cells on coverslips were incubated with 2.5 μg/ml BFA in HEPES-buffered normal medium, pH 7.2, without serum for 2 min at room temperature to induce the dissociation of βCOP from the Golgi. Rebinding of βCOP to the Golgi was assessed by washing cells four times with 1 ml of normal medium without serum, hypotonic medium (210 mOsm), or hypertonic medium (610 mOsm) and placing them at 37°C for 30 s. The average of the mean fluorescence intensity of βCOP staining was determined as in A. Vertical bars represent the SEM as determined from 25 measurements.

Figure 10

Golgi residents reemerge after long-term incubation in hypotonic medium, and reemergence is blocked by calphostin C. COS-7 cells, grown on 12-mm coverslips and either untreated (A–D) or pretreated with 100 nM calphostin C for 30 min (E–H), were removed from normal medium and placed in hypotonic (210-mOsm) medium (B–D) or hypotonic (210-mOsm) medium plus 100 nM calphostin C (F–H). Cells were fixed at 30 min (B and F), 3 h (C and G), or 6 h (D and H) and processed for immunofluorescence using a polyclonal antiserum against GM130. Cells maintained in normal medium in the absence (A) or presence of 100 nM calphostin C (E) were also fixed and stained.