(Received for publication, November 4, 1994; and in revised form, January 10, 1995)
From the
In the endocytic compartment, an acidic pH plays a key role in
receptor and ligand sorting, vesicular transport, and protein
degradation. In the secretory compartment, indirect estimates of trans-Golgi pH based on partitioning of weak bases and
following viral infection suggest a mildly acidic pH of >6.0. We
developed a liposome microinjection method to introduce fluorescent
indicators into the aqueous compartment of trans-Golgi in
living cells. In the presence of ATP and at 37 °C, 70-nm diameter
liposomes delivered their fluid-phase contents selectively into the trans-Golgi compartment as assessed by colocalization with the trans-Golgi stain N- {6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl}-sphingosine
(C-NBD-ceramide). Liposome fusion was ATP- and
temperature-dependent and blocked by N-ethylmaleimide but not
by guanosine 5`-O-(3-thiotriphosphate) (GTP
S). trans-Golgi pH in skin fibroblasts was 6.17 ± 0.02
(S.E., n = 174) as measured by ratio imaging confocal
microscopy using fluorescein and rhodamine-based indicators and an in vivo calibration procedure. trans-Golgi pH
increased to 6.8 ± 0.1 by cAMP agonists and to 6.5 ± 0.1
by protein kinase C activation. These results provide the first direct
measurement of trans-Golgi pH in living cells and demonstrate
pH regulation by second messengers.
It is well documented that a vacuolar-type proton pump maintains a relatively acidic pH in some organelles of the endosomal and secretory compartments(1, 2, 3) . An acidic pH relative to cytosol was demonstrated in trans-Golgi by accumulation of the lysosomotropic agent 3-(2,4-dinitroanilino)-3`-amino-N-methyl-dipropylamine(4, 5) . Two other lines of evidence suggest that trans-Golgi pH is >6.0, including analysis of pH-dependent fusion of viral spike proteins resulting in viral infectivity (6) and pH-dependent ligand binding to mannose 6-phosphate receptors(7) . A mildly acidic pH in trans-Golgi may play an important role in the transport of secretory proteins into secretory granules and in the post-translational processing of newly synthesized proteins(3, 8) .
Cytosolic pH has been studied extensively by fluorescence methods over the past decade with the development of a variety of pH-sensitive dyes and cell-trapable acetoxymethylester derivatives(9, 10) . New developments in fluorescence ratio imaging microscopy have enabled the mapping of pH in single cells (11) and in individual vesicles of the endosomal pathway(12, 13) . However, because the secretory compartments remain relatively inaccessible to fluid-phase fluorescent markers, there has been no method to selectively label in vivo the lumen of endoplasmic reticulum, Golgi, and secretory vesicles for direct measurement of pH.
We report here a novel method to deliver aqueous-phase fluorescent indicators into the lumen of the trans-Golgi compartment in living cells. Our strategy was inspired by a vesicle fusion method applied previously in semi-intact cells(14) , where liposomes were shown to fuse selectively with the trans-Golgi. Here, trans-Golgi in living human skin fibroblasts was labeled by cytoplasmic microinjection of 70-nm diameter liposomes containing membrane-impermeable fluorophores. A pH-sensitive fluorophore (fluorescein sulfonate) and a pH-insensitive fluorophore (sulforhodamine 101) were introduced into the trans-Golgi for direct measurement of pH by ratio imaging confocal microscopy. Average trans-Golgi pH was found to be 6.17 ± 0.02, and an unexpected regulatory effect of second messengers was demonstrated.
Normal human skin fibroblasts were microinjected with
uniform-sized liposomes of 70 ± 1 nm diameter containing
selected fluid-phase fluorescent probe(s). Liposomes containing 5
mM SR (a water-soluble fluorescent probe) fused with trans-Golgi and delivered their aqueous phase contents at 37
°C with a half-time of 15 min. Maximum trans-Golgi
fluorescence was observed at
30 min. Selective labeling of trans-Golgi was demonstrated with confocal microscopy by
colocalization of SR (red) with the specific trans-Golgi lipid-phase stain C
-NBD-ceramide (green) (15) (Fig. 1A).
Figure 1:
Selective labeling of the
lumen of the trans-Golgi compartment in normal human skin
fibroblasts. A, left: gallery of cells labeled with
the specific lipid phase trans-Golgi marker,
C-NBD-ceramide (5 µM C
-NBD-ceramide-BSA complex, 5 min, 37 °C); right: same cells microinjected with a suspension of 70-nm
diameter liposomes containing 5 mM sulforhodamine 101 in 2.5
mM ATP, 25 mM HEPES, 125 mM sucrose, 70
mM KCl, 2.5 mM MgCl
(pH 7.2) and
incubated for 30 min at 37 °C. B,
C
-NBD-ceramide image (left) and sulforhodamine 101
image (right) of a cell obtained after 0, 10, and 20 min of
incubation at 37 °C. C, representative cell microinjected
with the same liposome suspension but incubated at 23 °C. Scalebar, 2 µm; n, nucleus; arrow,
non-microinjected cell.
After a
30-min cell incubation at 37 °C, it was estimated that 30-50%
of microinjected liposomes fused with trans-Golgi (based on
the ratio of trans-Golgi specific to whole cell fluorescence
using liposomes containing the lipid phase marker TMR-PE). Additional
incubation at 37 °C resulted in a progressive decline in trans-Golgi fluorescence (Fig. 1B) due to a
combination of downstream and secretory traffic, and dye leakage. At 23
°C, however, labeling was stable for >60 min. Liposome fusion
was not detected when cells were incubated at 23 °C instead of at
37 °C after microinjection (Fig. 1C), and fusion
was inefficient (relative efficiency 0.2) when ATP was not
included in the microinjection buffer. The sensitivity of fusion
efficiency to added ATP may result from increased cytoplasmic ATP
concentration and/or from replacement of ATP loss associated with
microinjection. Fusion was completely blocked by addition to the
microinjection buffer of 5 mMN-ethylmaleimide but
not by up to 1 mM GTP
S, suggesting that liposome fusion
does not involve a GTP-dependent coating process(16) . Larger
(200-nm diameter) and smaller (<40-nm diameter, prepared by probe
sonication) liposomes did not fuse efficiently. Although we do not know
the precise mechanism by which trans-Golgi is labeled
selectively, the results above suggest that the liposomes introduced by
microinjection may be misrecognized by the trans-Golgi as
70-nm diameter transport vesicles arising from an earlier
compartment(16) .
For measurement of pH, the combination of
SR (pH insensitive, red fluorescence) and FS (pK
6.3, green fluorescence) was chosen based on their bright
fluorescence, self-quenching at high concentrations(17) ,
non-overlapping fluorescence spectra, optimal pK
,
and low membrane permeability(10, 18) . After
microinjection of liposomes containing SR and FS and a 30-min
incubation at 37 °C, pairs of confocal images were recorded by a
cooled CCD camera (Fig. 2A). trans-Golgi pH was
calculated by quantitative image analysis from the FS-to-SR signal
ratio after background subtraction. Absolute pH determination required in situ calibration of trans-Golgi FS-to-SR signal
ratio versus pH. trans-Golgi pH was set equal to
extracellular pH using bafilomycin A
(proton pump
inhibitor, 10 nM) and the ionophore pair monensin (Na-H
exchanger, 10 µM) + CCCP (protonophore, 1
µM) (Fig. 2B). The FS-to-SR signal ratio
did not change when the concentrations of bafilomycin A
,
monensin, and/or CCCP were increased by 3-fold or when incubation with
ionophores was extended to 30 min. Monensin in the presence of
bafilomycin A
did not affect Golgi structure. Nigericin
(K-H exchanger, 1 µM) could not be used for in situ calibration because it disrupted the Golgi structure, similar to
results obtained using the Golgi-disrupting agents brefeldin A (5
µg/ml) and nocodazole (20 µg/ml). The apparent
pK
of 6.3 measured in situ (Fig. 2C) was identical to that in cell-free
aqueous solution. Time course studies indicated <5% indicator
photobleaching or leakage occurred in 30 min under the conditions of
our experiment.
Figure 2: trans-Golgi pH in normal human skin fibroblasts measured at 23 °C. Cells were microinjected with liposomes containing 5 mM SR and 30 mM FS and incubated at 37 °C for 30 min. A, colocalization of FS (left, green) and SR (middle, red); right, pseudocolored ratio image of FS/SR after background subtraction. B, FS, SR, and FS/SR images of a cell after perfusion for 10 min with calibration buffer at pH 6.2. C, in situ calibration curve of FS/SR signal ratio versus pH.
The FS-to-SR signal ratio was 0.48 ± 0.02
(S.E.) in 174 skin fibroblasts, corresponding to a trans-Golgi
pH of 6.17 ± 0.02 (Fig. 2C). There was little pH
variation in the lumen of the trans-Golgi compartment as shown
by the representative pseudocolored ratio image in Fig. 2, right. Analysis of pH distributions obtained from separate
cells indicated that mean trans-Golgi pH in 75% of cells was
in the range 6.0-6.3. It is noted that the total intraliposomal
volume microinjected into each cell (4
10
cm
) was much smaller than cell volume (
5.2
10
cm
) or estimated Golgi
volume (4
10
cm
(19) ).
Taken together with the incomplete fusion efficiency (30-50%) and
the low intraliposomal buffer capacity (
6 mM/pH unit at
pH 7.0) compared with that in Golgi (50 mM/pH unit, measured
by NH
Cl pulse technique, see Fig. 3), it is unlikely
that the liposome fusion process affects trans-Golgi pH.
Figure 3:
Effect
of regulatory factors on trans-Golgi pH. Measurements were
performed in microinjected cells after 30 min at 37 °C and an
additional 30 min in PBS containing indicated compounds at 23 °C.
Concentrations: 30 mM NHCl, 10 nM bafilomycin A
, 10 nM platelet-derived growth
factor B/B (PDGF), 1 µM phorbol 12-myristate
13-acetate (PMA), 0.5 mM CPT-cAMP.
Cl
-free buffer indicates replacement of
Cl
by the membrane-impermeant anion
isethionate.
The influence of putative regulators of trans-Golgi pH was
investigated (Fig. 3). As anticipated, inhibition of the
vacuolar proton pump by bafilomycin A or addition of a weak
base (NH
Cl) caused trans-Golgi alkalinization. trans-Golgi pH was mildly increased by protein kinase C
activation by phorbol 12-myristate 13-acetate, and platelet-derived
growth factor, whereas protein kinase A activation by forskolin or a
cell-permeable cAMP analog (CPT-cAMP) remarkably elevated trans-Golgi pH to 6.8 ± 0.1. Interestingly, a smaller
but significant cAMP-induced alkalinization was reported in early
endosomes from Swiss 3T3 fibroblasts labeled with a fluorescent
transferrin(12) . To determine whether intracellular
cAMP-stimulated Cl
channels (20, 21, 22) were responsible for the
alkalinization, experiments were performed in which Cl
was replaced by the impermeant anion isethionate. Cytosolic
Cl
activity decreased from 55 to <5 mM by
this maneuver as measured by SPQ fluorescence(23) .
Cl
removal itself caused a small trans-Golgi
alkalinization but did not abolish the large cAMP-dependent
alkalinization.
Our results establish an effective method to label the aqueous phase of trans-Golgi in living cells and provide the first direct measurement of trans-Golgi pH. The acidic lumenal pH is consistent with the identification of multiple pH-dependent events in the secretory pathway, including the sorting and storage of numerous secretory proteins(3, 24, 25, 26) , aggregation of pancreatic secretory proteins(27) , protein post-translational modifications by sialyltransferases(26) , binding of KDEL to its receptor(28) , and activation of virus fusion protein(7) . In addition, recent experiments on permeabilized cells suggest that pH between 6 and 6.2 in the trans-Golgi is optimal for enzymatic cleavage of prosomatostatin(29) .
The introduction by liposome fusion of fluorescent indicators of calcium, monovalent ions, and membrane potential should enable measurements of trans-Golgi ion activities and mechanisms of protein processing and secretion. The strong alkalinization of trans-Golgi by cAMP agonists is an unexpected finding that may provide an explanation, when taken together with recent data on effects of lumenal pH on vesicular transport(30) , for the cAMP-dependent inhibition of vesicular transport in some cell types (31) . Last, the ability to quantify trans-Golgi pH should facilitate direct examination of the ``defective organelle acidification hypothesis'' proposed to be the cellular basis of cystic fibrosis(32) .