Cytoskeleton: i t s Role in Cellular Function
41. Drubin, L). G., Feinstein, S. C.. Shooter. E. M. & Kirschner, M. W. (1985)J. Cell Hiol. 101. 1799-1807 42. Hrugg. H. & Matus. A. (1988) J. Cell Hiol. 107. 643-650 43. Diaz-Nido. J., Serrano, I,.. Mendez. E. & Avila, J. (1988)J. Cell Hiol. 106,2057-2065 44. Viereck, C. & Matus, A. (1990) Hrain Kes. 508, 257-264 45. Fischer, I. & Komano-Clarke, G. (1990) J. Neurochem. 55.328-333
46. Sato-Yoshitake, K., Shiomura. Y.. Miyasaka, H. & Hirokawa, N. (1989) Neuron 3,229-238 47. Hasegawa, M.. Arai, T. & Ihara, Y. (1 990) Neuron 4, 909-9 18 48. Gordon-Weeks, 1’. R. (1988) Electron Microsc. Rev. 1,201-219
Keceived 7 June 1901
~
Calcium, the cytoskeleton and calpactin (annexin II) in exocytotic secretion from adrenal chromaffin and mammary epithelial cells Robert D. Burgoyne,*t Susan E. Handel,* Alan Morgan,* Michelle E. Rennison,* Mark D. Turner* and Colin J. Wilde$ *Department of Physiology, University of Liverpool, PO Box 147, Liverpool L69 3BX, U. K. and $Hannah Research Institute, Ayr KA6 SHL, U.K.
Introduction Exocytosis occurs in virtually all cell types; it can occur in a regulated fashion such that stored secretory vesicles only undergo exocytosis in response to changes in intracellular second messengers such as a rise in cytosolic free calcium concentration ([Ca2+],)or in a constitutive fashion immediately following vesicle biogenesis [ 11. A great deal is now known about the intracellular signals that activate exocytosis in regulated secretory cells such as neurons, endocrine and exocrine cells, but the molecular mechanisms involved in exocytotic membrane fusion are still unclear. It is not known whether a single exocytotic mechanism exists in all cell types or whether a variety of mechanisms are expressed depending on the physiological function of the particular cell. In addition, the degree of similarity between regulated and constitutive exocytosis is unknown. W e have begun to compare the features of exocytosis in bovine adrenal chromaffin cells, in which catecholamine secretion is activated only after an increase in [Ca”], [2], with that in lactating mouse mammary epithelial cells, in which proteins are apparently secreted immediately following synthesis in a constitutive manner [ 31.
Calcium and exocytosis in chromaffin and mammary epithelial cells In adrenal chromaffin cells, catecholamines and other secretory products are stored in dense-core secretory vesicles (chromaffin granules) for prolonged periods and are only secreted following cell
t T o whom correspondence should be addressed
stimulation. The major signal for exocytosis in these cells is a rise in [Ca’+], following stimulation of Caz+ entry by a variety of agonists. Exocytosis can be directly activated in permeabilized chromaffin cells simply by directly raising [CaL+],,but it is regulated by other factors such as protein kinase C activation (reviewed in [2]). In the case of mammary epithelial cells, secretory proteins such as the caseins are packaged into dense-core secretory vesicles and are released from the cells after a lag period of only around 1 h after synthesis [ 3 , 41. This is consistent with constitutive secretion immediately after processing of the newly synthesized secretory proteins, movement through the early stages of the secretory pathway and packaging into secretory vesicles. No known external signals are required to elicit exocytotic secretion from these cells in vivo or in vitro, but secretion is under inhibitory autocrine control [S]. W e have found that measures that would be expected to reduce the basal [Ca”], have no effect on the extent of constitutive secretion of pre-synthesized protein from lactating mammary cells, indicating that basal exocytotic secretion is independent of [Ca’+], [4]. Treatment of cells with the calcium ionophore ionomycin, however, resulted in a 2-fold increase in the extent of protein secretion. This effect of ionomycin was abolished if excess EGTA was also present indicating that it was CaLf dependent. In addition, it was found that ionomycin was able to stimulate exocytosis at a time after constitutive exocytosis had terminated. These data are consistent with the existence of separate constitutive and regulated pathways for protein secretion in mammary cells [ 41.
1991
I085
Biochemical Society Transactions
60 -
The cytoskeleton and exocytosis in chromaffin and mammary epithelial cells
T
50 -
I086 40
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30 20
-
10
-
00
30
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45
60
Time (s) Control
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In unstimulated adrenal chromaffin cells, most of the secretory granules are excluded from a 200 nmwide zone at the cell periphery [ h ] ;this is the site of the cortical actin network. The appearance of the unstimulated cells in the electron microscope suggests that the secretory granules are prevented from reaching exocytotic sites on the plasma membrane by the cortical cytoskeleton and work over the past few years has suggested that disassembly of the cortical actin network after cell stimulation is necessary for a secretory response [7]. Early ideas that secretion in chromaffin cells would require active transport of secretory vesicles to the plasma membrane after cell activation have not received support from experimental data. Microtubules may be involved in transport of newly formed chromaftin granules out from the Golgi region of the cell, but do not appear to be necessary for exocytosis [7]. In addition, intact actin filaments also do not appear to be required for exocytosis in chromaffin cells. In contrast, stimulation of chrornaffin cells in a variety of ways results in a disassembly or reorganization of the cortical actin network that can be detected using either biochemical assays or rhodamine-phalloidin staining [ 7, 8- 10 1. Figure 1(a ) shows the transient changes in actin that occur after nicotinic stimulation using either the 1)NAase I
I
Nocodazole
0
I )
I
I
I
2
1
I
I
I
3
4
5
( a ) The extent of actin assembly in bovine chromaffin cells was assessed using either the DNAase I inhibition assay for F-actin
Time (h)
[8] or an assay that determined the amount of actin associated with the Triton X-100-insoluble cytoskeleton (Cyto actin) [ I I ] Both assays demonstrated a transient reduction in assembled actin in response to stimulation of chromaffin cells in suspension with 10 pwnicotine ( b ) Effect of the microtubuledepolymerizing drug nocodazole on secretion from lactating mouse mammary epithelial cells Protein secretion from isolated lactating mammary epithelial cells was determined in the absence or presence of I pg of nocodazoleiml Isolated acini from a 10 day, post-partum mouse were incubated with 10 pCi/mI [3sS]methioninewith or without nocodazole and the extent of secretion at various times determined by measuring released trichloroacetic acid-precipitable counts (c) Effect of treatment with 10 pM-cytochalasin D t o disassemble cortical actin on protein secretion from isolated lactating mouse mammary acini The mammary cells were pulse-labelled for I h with 25 p C i of [35S]methionine/ml,washed and incubated with ( a ) or without (A)cytochalasin D As a measure of protein secretion, the released trichloroacetic acid-precipitable counts are expressed as a percentage of the total counts incorporated into trichloroacetic-precipitable protein Cytochalasin D treatment did not stimulate exocytosis in these cells [4]
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Volume 19
Actin and microtubules in exocytotic secretion from adrenal chromaffin cells and mammary epithelial cells
Time (h)
40
1
Fig. I
I
Cytoskeleton: its Role in Cellular Function
inhibition assay or an assay based on quantification of the amount of actin associated with the Triton X- 100-insoluble cytoskeleton. Despite the fact that the two assays shown in Fig. l ( a ) measure different aspects of actin organization, the results are similar. Khodamine-phalloidin staining to visualize assembled actin filaments shows that assembled actin is concentrated at the cell periphery and, after stimulation, staining is either lost around the whole cell [ X I or in patches that coincide with the sites of exocytosis [9]. The reorganization of actin appears to involve both Ca' -dependent and Ca'+-independent mechanisms [ 1 11. Under certain conditions, not involving a rise in [ Ca"],, actin disassembly is not accompanied by extensive exocytosis [ 111, and this indicates that the removal of the actin barrier is not itself sufficient to elicit exocytosis in chromaffin cells and that Ca" is also required for the final membrane fusion step in exocytosis after the secretory granules reach the plasma membrane. The involvement of the cytoskeleton in secretion from lactating mammary epithelial cells appears to be very different from that in adrenal chromaffin cells. There is growing evidence on the importance of microtubules for organelle movement in cells [ 121, including movement of constitutive secretory vesicles in polarized epithelia [ 131. It has been shown that treatment of mammary cells with colchicine to disassemble microtubules results in an accumulation of secretory vesicles consistent with an inhibition of exocytosis [ 14-16]. However, rather high concentrations of colchicine were often used and toxic side-effects could not be ruled out. For this reason we have examined the effect of the more potent anti-microtubule drug nocodazole on secretion from freshly isolated acini from lactating mouse mammary gland. Nocodazole had no effect on the extent of protein synthesis in mammary cells, based on the incorporation of [ "'Slmethionine into trichloroacetic acid-precipitable protein, but abolished protein secretion (Fig. lb). No cellular damage was detected by electron microscopy in nocodazole-treated cells. Another difference between mammary and chromaffin cells is that the cortical actin cytoskeleton in mammary cells does not appear to act as a barrier as is the case in chromaffin cells. The effect of treatment of mammary cells with cytochalasin B has previously been studied [ 161, and we have examined the effect of the more-specific drug cytochalasin D, since there is a potential problem with inhibition of glucose uptake and hence inhibition of energydependent cell function, by cytochalasin H. A cortical actin network in isolated lactating mammary +
cells can be visualized using rhodamine-phalloidin staining and it was found to disassemble after treatment with cytochalasin D. After long-term treatment with the drug, punctate staining was seen presumably due to the appearance of actin filament accumulations. Under these conditions, despite the removal of any potential inhibitory effect of the cortical cytoskeleton, protein secretion is not enhanced over control levels (Fig. lc) [4].These results suggest that in mammary cells the cortical actin network does not act as a barrier to exocytosis. This interpretation would suggest that the ionomycin-induced stimulation of regulated protein secretion from mammary cells by increased [CaL+], is not due to an effect on the cortical actin network. It was previously suggested, after the observation that both regulated and constitutive exocytosis can occur in the same cell type, that regulated exocytotic vesicles may be specifically impeded by the cortical actin network with constitutive secretory vesicles free to diffuse to the plasma membrane owing to a lack of interactions between vesicle components and actin filaments [ 171. Our examination of secretion from mammary cells suggests that in this cell type neither constitutive nor regulated secretory vesicles are impeded by the actin network. In support of this conclusion, we found from electron microscopical examination of Triton X-100-extracted ghosts, that relatively few actin filaments were present in the cortical region of the cells (Fig. 2).
Calpactin (annexin II) and exocytosis in adrenal chromaffin cells and mammary epithelial cells It is not clear what the targets are for the action of in . activating exocytosis in adrenal chromaffin cells and stimulating the Ca'+-regulated pathway in mammary cells. Among the possible target proteins are protein kinase C and the calcium-, phospholipid- and actin-binding protein calpactin (annexin 11). Protein kinase C regulates CaL+dependent secretion in chromaffin cells, but does not appear to be important in mammary cells, since activation of protein kinase C with phorbol esters has no effect on the extent of protein secretion. Calpactin is one of a family of proteins isolated in several laboratories on the basis of their ability to bind in a Calf-dependent manner to membranes or to Triton X- 100-insoluble cytoskeletons. Early work found that calpactin only associated with actin at Ca" concentrations of 0.5-1 mai. which called into question the possibility that this protein could associate with actin at physiological CaL' levels.
cal+
1991
1087
Biochemical Society Transactions
Fig. 2
Organization of the cytoskeleton in Triton X- IOOextracted ghosts of lactating mammary epithelial cells
I088
Mammary cells isolated from the lactating mouse mammary gland were extracted by resuspending cells in 2% (v/v) Triton x-100 in 20 mM-NaH,PO,, 100 mM-KCI, I 5 mM-MgCI,, I m w EGTA, I M-glycerol, 0 5% ( w h ) bovine serum albumin, pH 7 I for 5 min The cells were then fixed by addition of an equal volume of double-strength fixative [4% (v/v) glutaraldehyde, in 75 mn-phosphate buffer, pH 74, with 100 mM L-lysine and 10 m~-EGTA]This fixative and subsequent steps were chosen to preserve actin filaments The cells were pelleted and fixed further in 2% ( v h ) glutaraldehyde in 75 mn-phosphate buffer, pH 7 4 for I h After several cold washes the pellets were postfixed in I % (w/v) osmium tetroxide for 15 min on ice and further processed for electron microscopy The micrographs show examples of well-preserved ghosts with cytoskeletal elements often with bound ribosomes In ( a ) the cell shown had one region of cortex containing unusually high levels of actin filaments (arrowheads) chosen to show that actin filaments were preserved by the method used More typically relatively few actin filaments were seen in the rest of the cell cortex In ( b ) an extracted acinus is shown with adherens junctions between cells [large arrows in ( b ) ] and extracted microvilli (arrowheads) in the lumen (L) The luminal cell cortex contains few actin filaments Microtubules (open arrow) were often seen near the cell cortex Scale bars, I ,um
a
1
.
More recently, it has been established that calpactin. and in particular the heterotetrameric form of this protein, can bundle actin filaments in a Ca'+dependent nianner over micromolar Ca'+ concentrations raising the possibility that in addition to its association with the plasma membrane in cells, calpactin could also be a bona tide actin-binding protein [ 181. Moreover, it was suggested that its binding to membranes such as the chromaffin
Volume 19
granule membrane may be due to the presence of actin on these membranes. In studies on the organization of calpactin in intact cells, the protein has been found to be concentrated on the inner surface of the plasma membrane, where, in the presence of micromolar CaL+,it may be able to form short filaments that cross-link plasma and granule membranes [ 191. Calpactin may play a role in membrane fusion in exocytosis in adrenal chromaffin cells since it is able to bind to, aggregate and stimulate fusion of chromaffin granules in vitro [20]. As noted above, in intact chromaffin cells much of the calpactin is found on the plasma membrane, but some calpactin is also present on isolated chromaffin granule membranes in a form that can not be removed by EGTA 1213. Calpactin was the only annexin found to be associated with chromaffin granules, thus implicating this particular annexin in exocytosis [21]. From studies on digitonin-permeabilized chromaffin cells evidence has been found suggesting that calpactin may be one of the proteins required for Ca'+dependent exocytosis in this cell type [22-241. For example, calpactin is able to maintain the responsiveness of permeabilized cells losing secretory competence owing to leakage of cytosolic protein components. It is not clear whether calpactin is acting as a membrane fusogen or instead as a membrane cross-linker to anchor chromaffin granules close to the plasma membrane. W e have examined the expression of calpactin in mouse mammary cells developing in vivo using immunocytochemical staining with an anti-peptide antibody directed against the N-terminal 15 amino acids of calpactin [25]. In tissue taken during pregnancy, calpactin had a diffuse general distribution in cell types in the mammary gland. In contrast, in post-partum, lactating mammary gland, a rnarked concentration of calpactin staining was seen in mammary epithelial cells on the apical (secretory) membrane (Fig. 3). Calpactin appears, therefore, to redistribute to the secretory pole of the cells after differentiation of the cells and the final achievement of the secretory phenotype [25]. Calpactin was also detected at the sites of compound exocytosis (Fig. 31)) and on casein secretory vesicles (Fig. 3E). It still remains to be determined whether calpactin plays a merely structural role in lactating marnniary cells or whether it is involved in constitutive and/or regulated exocytosis. It is intriguing that in secreting mammary cells the casein-containing secretory vesicles were found to be linked to the plasma membrane before fusion by short cross-links reminiscent of those formed by calpactin bound to
Cytoskeleton: its Role in Cellular Function
Fig. 3 lmmunoperoxidase localization of calpactin (annexin II) in lactating mouse mammary gland Vibratome sections of 10 day, post-partum mouse mammary gland were stained with anti-cal ( I I5), processed using the immunoperoxidase technique and prepared for electron microscopy [25] (A) Dark reaction product is present along the apical membranes but, not the basal (arrowheads) membranes of mammary epithelial cells (B) Unstained apical region of cell (C) Stained apical region of mammary cell showing plasma membrane and microvillar staining Secretory vesicle membranes and sites of compound exocytosis were also stained by the antiserum (arrows in D and E) Scale bars, I p m
I089
-
lipid vesicles and observed in stimulated chromaffin cells [U]. The results summarized here have allowed us to begin to compare exocytotic secretion in two very different cell types. Continued characterization of adrenal chromaffin and mammary cells should give further insight into the similarities and differences between the regulated and constitutive exocytotic pathways.
0 . Hurgoyne, K. I)., (;eisow. 51. J. 81 Harron, J. (1982) I’roc. K. Soc. Idondon H 216, 1 1 1-1 15 7. Cheek, 7’. K. & Ihrgoyne. K.L). (1991) The Neuronal Cytoskeleton (Ihirgoync. I
41. Drubin, L). G., Feinstein, S. C.. Shooter. E. M. & Kirschner, M. W. (1985)J. Cell Hiol. 101. 1799-1807 42. Hrugg. H. & Matus. A. (1988) J. Cell Hiol. 107. 643-650 43. Diaz-Nido. J., Serrano, I,.. Mendez. E. & Avila, J. (1988)J. Cell Hiol. 106,2057-2065 44. Viereck, C. & Matus, A. (1990) Hrain Kes. 508, 257-264 45. Fischer, I. & Komano-Clarke, G. (1990) J. Neurochem. 55.328-333
46. Sato-Yoshitake, K., Shiomura. Y.. Miyasaka, H. & Hirokawa, N. (1989) Neuron 3,229-238 47. Hasegawa, M.. Arai, T. & Ihara, Y. (1 990) Neuron 4, 909-9 18 48. Gordon-Weeks, 1’. R. (1988) Electron Microsc. Rev. 1,201-219
Keceived 7 June 1901
~
Calcium, the cytoskeleton and calpactin (annexin II) in exocytotic secretion from adrenal chromaffin and mammary epithelial cells Robert D. Burgoyne,*t Susan E. Handel,* Alan Morgan,* Michelle E. Rennison,* Mark D. Turner* and Colin J. Wilde$ *Department of Physiology, University of Liverpool, PO Box 147, Liverpool L69 3BX, U. K. and $Hannah Research Institute, Ayr KA6 SHL, U.K.
Introduction Exocytosis occurs in virtually all cell types; it can occur in a regulated fashion such that stored secretory vesicles only undergo exocytosis in response to changes in intracellular second messengers such as a rise in cytosolic free calcium concentration ([Ca2+],)or in a constitutive fashion immediately following vesicle biogenesis [ 11. A great deal is now known about the intracellular signals that activate exocytosis in regulated secretory cells such as neurons, endocrine and exocrine cells, but the molecular mechanisms involved in exocytotic membrane fusion are still unclear. It is not known whether a single exocytotic mechanism exists in all cell types or whether a variety of mechanisms are expressed depending on the physiological function of the particular cell. In addition, the degree of similarity between regulated and constitutive exocytosis is unknown. W e have begun to compare the features of exocytosis in bovine adrenal chromaffin cells, in which catecholamine secretion is activated only after an increase in [Ca”], [2], with that in lactating mouse mammary epithelial cells, in which proteins are apparently secreted immediately following synthesis in a constitutive manner [ 31.
Calcium and exocytosis in chromaffin and mammary epithelial cells In adrenal chromaffin cells, catecholamines and other secretory products are stored in dense-core secretory vesicles (chromaffin granules) for prolonged periods and are only secreted following cell
t T o whom correspondence should be addressed
stimulation. The major signal for exocytosis in these cells is a rise in [Ca’+], following stimulation of Caz+ entry by a variety of agonists. Exocytosis can be directly activated in permeabilized chromaffin cells simply by directly raising [CaL+],,but it is regulated by other factors such as protein kinase C activation (reviewed in [2]). In the case of mammary epithelial cells, secretory proteins such as the caseins are packaged into dense-core secretory vesicles and are released from the cells after a lag period of only around 1 h after synthesis [ 3 , 41. This is consistent with constitutive secretion immediately after processing of the newly synthesized secretory proteins, movement through the early stages of the secretory pathway and packaging into secretory vesicles. No known external signals are required to elicit exocytotic secretion from these cells in vivo or in vitro, but secretion is under inhibitory autocrine control [S]. W e have found that measures that would be expected to reduce the basal [Ca”], have no effect on the extent of constitutive secretion of pre-synthesized protein from lactating mammary cells, indicating that basal exocytotic secretion is independent of [Ca’+], [4]. Treatment of cells with the calcium ionophore ionomycin, however, resulted in a 2-fold increase in the extent of protein secretion. This effect of ionomycin was abolished if excess EGTA was also present indicating that it was CaLf dependent. In addition, it was found that ionomycin was able to stimulate exocytosis at a time after constitutive exocytosis had terminated. These data are consistent with the existence of separate constitutive and regulated pathways for protein secretion in mammary cells [ 41.
1991
I085
Biochemical Society Transactions
60 -
The cytoskeleton and exocytosis in chromaffin and mammary epithelial cells
T
50 -
I086 40
-
30 20
-
10
-
00
30
I5
45
60
Time (s) Control
5
4
3
2
In unstimulated adrenal chromaffin cells, most of the secretory granules are excluded from a 200 nmwide zone at the cell periphery [ h ] ;this is the site of the cortical actin network. The appearance of the unstimulated cells in the electron microscope suggests that the secretory granules are prevented from reaching exocytotic sites on the plasma membrane by the cortical cytoskeleton and work over the past few years has suggested that disassembly of the cortical actin network after cell stimulation is necessary for a secretory response [7]. Early ideas that secretion in chromaffin cells would require active transport of secretory vesicles to the plasma membrane after cell activation have not received support from experimental data. Microtubules may be involved in transport of newly formed chromaftin granules out from the Golgi region of the cell, but do not appear to be necessary for exocytosis [7]. In addition, intact actin filaments also do not appear to be required for exocytosis in chromaffin cells. In contrast, stimulation of chrornaffin cells in a variety of ways results in a disassembly or reorganization of the cortical actin network that can be detected using either biochemical assays or rhodamine-phalloidin staining [ 7, 8- 10 1. Figure 1(a ) shows the transient changes in actin that occur after nicotinic stimulation using either the 1)NAase I
I
Nocodazole
0
I )
I
I
I
2
1
I
I
I
3
4
5
( a ) The extent of actin assembly in bovine chromaffin cells was assessed using either the DNAase I inhibition assay for F-actin
Time (h)
[8] or an assay that determined the amount of actin associated with the Triton X-100-insoluble cytoskeleton (Cyto actin) [ I I ] Both assays demonstrated a transient reduction in assembled actin in response to stimulation of chromaffin cells in suspension with 10 pwnicotine ( b ) Effect of the microtubuledepolymerizing drug nocodazole on secretion from lactating mouse mammary epithelial cells Protein secretion from isolated lactating mammary epithelial cells was determined in the absence or presence of I pg of nocodazoleiml Isolated acini from a 10 day, post-partum mouse were incubated with 10 pCi/mI [3sS]methioninewith or without nocodazole and the extent of secretion at various times determined by measuring released trichloroacetic acid-precipitable counts (c) Effect of treatment with 10 pM-cytochalasin D t o disassemble cortical actin on protein secretion from isolated lactating mouse mammary acini The mammary cells were pulse-labelled for I h with 25 p C i of [35S]methionine/ml,washed and incubated with ( a ) or without (A)cytochalasin D As a measure of protein secretion, the released trichloroacetic acid-precipitable counts are expressed as a percentage of the total counts incorporated into trichloroacetic-precipitable protein Cytochalasin D treatment did not stimulate exocytosis in these cells [4]
T
Control
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30
20
10
0
Volume 19
Actin and microtubules in exocytotic secretion from adrenal chromaffin cells and mammary epithelial cells
Time (h)
40
1
Fig. I
I
Cytoskeleton: its Role in Cellular Function
inhibition assay or an assay based on quantification of the amount of actin associated with the Triton X- 100-insoluble cytoskeleton. Despite the fact that the two assays shown in Fig. l ( a ) measure different aspects of actin organization, the results are similar. Khodamine-phalloidin staining to visualize assembled actin filaments shows that assembled actin is concentrated at the cell periphery and, after stimulation, staining is either lost around the whole cell [ X I or in patches that coincide with the sites of exocytosis [9]. The reorganization of actin appears to involve both Ca' -dependent and Ca'+-independent mechanisms [ 1 11. Under certain conditions, not involving a rise in [ Ca"],, actin disassembly is not accompanied by extensive exocytosis [ 111, and this indicates that the removal of the actin barrier is not itself sufficient to elicit exocytosis in chromaffin cells and that Ca" is also required for the final membrane fusion step in exocytosis after the secretory granules reach the plasma membrane. The involvement of the cytoskeleton in secretion from lactating mammary epithelial cells appears to be very different from that in adrenal chromaffin cells. There is growing evidence on the importance of microtubules for organelle movement in cells [ 121, including movement of constitutive secretory vesicles in polarized epithelia [ 131. It has been shown that treatment of mammary cells with colchicine to disassemble microtubules results in an accumulation of secretory vesicles consistent with an inhibition of exocytosis [ 14-16]. However, rather high concentrations of colchicine were often used and toxic side-effects could not be ruled out. For this reason we have examined the effect of the more potent anti-microtubule drug nocodazole on secretion from freshly isolated acini from lactating mouse mammary gland. Nocodazole had no effect on the extent of protein synthesis in mammary cells, based on the incorporation of [ "'Slmethionine into trichloroacetic acid-precipitable protein, but abolished protein secretion (Fig. lb). No cellular damage was detected by electron microscopy in nocodazole-treated cells. Another difference between mammary and chromaffin cells is that the cortical actin cytoskeleton in mammary cells does not appear to act as a barrier as is the case in chromaffin cells. The effect of treatment of mammary cells with cytochalasin B has previously been studied [ 161, and we have examined the effect of the more-specific drug cytochalasin D, since there is a potential problem with inhibition of glucose uptake and hence inhibition of energydependent cell function, by cytochalasin H. A cortical actin network in isolated lactating mammary +
cells can be visualized using rhodamine-phalloidin staining and it was found to disassemble after treatment with cytochalasin D. After long-term treatment with the drug, punctate staining was seen presumably due to the appearance of actin filament accumulations. Under these conditions, despite the removal of any potential inhibitory effect of the cortical cytoskeleton, protein secretion is not enhanced over control levels (Fig. lc) [4].These results suggest that in mammary cells the cortical actin network does not act as a barrier to exocytosis. This interpretation would suggest that the ionomycin-induced stimulation of regulated protein secretion from mammary cells by increased [CaL+], is not due to an effect on the cortical actin network. It was previously suggested, after the observation that both regulated and constitutive exocytosis can occur in the same cell type, that regulated exocytotic vesicles may be specifically impeded by the cortical actin network with constitutive secretory vesicles free to diffuse to the plasma membrane owing to a lack of interactions between vesicle components and actin filaments [ 171. Our examination of secretion from mammary cells suggests that in this cell type neither constitutive nor regulated secretory vesicles are impeded by the actin network. In support of this conclusion, we found from electron microscopical examination of Triton X-100-extracted ghosts, that relatively few actin filaments were present in the cortical region of the cells (Fig. 2).
Calpactin (annexin II) and exocytosis in adrenal chromaffin cells and mammary epithelial cells It is not clear what the targets are for the action of in . activating exocytosis in adrenal chromaffin cells and stimulating the Ca'+-regulated pathway in mammary cells. Among the possible target proteins are protein kinase C and the calcium-, phospholipid- and actin-binding protein calpactin (annexin 11). Protein kinase C regulates CaL+dependent secretion in chromaffin cells, but does not appear to be important in mammary cells, since activation of protein kinase C with phorbol esters has no effect on the extent of protein secretion. Calpactin is one of a family of proteins isolated in several laboratories on the basis of their ability to bind in a Calf-dependent manner to membranes or to Triton X- 100-insoluble cytoskeletons. Early work found that calpactin only associated with actin at Ca" concentrations of 0.5-1 mai. which called into question the possibility that this protein could associate with actin at physiological CaL' levels.
cal+
1991
1087
Biochemical Society Transactions
Fig. 2
Organization of the cytoskeleton in Triton X- IOOextracted ghosts of lactating mammary epithelial cells
I088
Mammary cells isolated from the lactating mouse mammary gland were extracted by resuspending cells in 2% (v/v) Triton x-100 in 20 mM-NaH,PO,, 100 mM-KCI, I 5 mM-MgCI,, I m w EGTA, I M-glycerol, 0 5% ( w h ) bovine serum albumin, pH 7 I for 5 min The cells were then fixed by addition of an equal volume of double-strength fixative [4% (v/v) glutaraldehyde, in 75 mn-phosphate buffer, pH 74, with 100 mM L-lysine and 10 m~-EGTA]This fixative and subsequent steps were chosen to preserve actin filaments The cells were pelleted and fixed further in 2% ( v h ) glutaraldehyde in 75 mn-phosphate buffer, pH 7 4 for I h After several cold washes the pellets were postfixed in I % (w/v) osmium tetroxide for 15 min on ice and further processed for electron microscopy The micrographs show examples of well-preserved ghosts with cytoskeletal elements often with bound ribosomes In ( a ) the cell shown had one region of cortex containing unusually high levels of actin filaments (arrowheads) chosen to show that actin filaments were preserved by the method used More typically relatively few actin filaments were seen in the rest of the cell cortex In ( b ) an extracted acinus is shown with adherens junctions between cells [large arrows in ( b ) ] and extracted microvilli (arrowheads) in the lumen (L) The luminal cell cortex contains few actin filaments Microtubules (open arrow) were often seen near the cell cortex Scale bars, I ,um
a
1
.
More recently, it has been established that calpactin. and in particular the heterotetrameric form of this protein, can bundle actin filaments in a Ca'+dependent nianner over micromolar Ca'+ concentrations raising the possibility that in addition to its association with the plasma membrane in cells, calpactin could also be a bona tide actin-binding protein [ 181. Moreover, it was suggested that its binding to membranes such as the chromaffin
Volume 19
granule membrane may be due to the presence of actin on these membranes. In studies on the organization of calpactin in intact cells, the protein has been found to be concentrated on the inner surface of the plasma membrane, where, in the presence of micromolar CaL+,it may be able to form short filaments that cross-link plasma and granule membranes [ 191. Calpactin may play a role in membrane fusion in exocytosis in adrenal chromaffin cells since it is able to bind to, aggregate and stimulate fusion of chromaffin granules in vitro [20]. As noted above, in intact chromaffin cells much of the calpactin is found on the plasma membrane, but some calpactin is also present on isolated chromaffin granule membranes in a form that can not be removed by EGTA 1213. Calpactin was the only annexin found to be associated with chromaffin granules, thus implicating this particular annexin in exocytosis [21]. From studies on digitonin-permeabilized chromaffin cells evidence has been found suggesting that calpactin may be one of the proteins required for Ca'+dependent exocytosis in this cell type [22-241. For example, calpactin is able to maintain the responsiveness of permeabilized cells losing secretory competence owing to leakage of cytosolic protein components. It is not clear whether calpactin is acting as a membrane fusogen or instead as a membrane cross-linker to anchor chromaffin granules close to the plasma membrane. W e have examined the expression of calpactin in mouse mammary cells developing in vivo using immunocytochemical staining with an anti-peptide antibody directed against the N-terminal 15 amino acids of calpactin [25]. In tissue taken during pregnancy, calpactin had a diffuse general distribution in cell types in the mammary gland. In contrast, in post-partum, lactating mammary gland, a rnarked concentration of calpactin staining was seen in mammary epithelial cells on the apical (secretory) membrane (Fig. 3). Calpactin appears, therefore, to redistribute to the secretory pole of the cells after differentiation of the cells and the final achievement of the secretory phenotype [25]. Calpactin was also detected at the sites of compound exocytosis (Fig. 31)) and on casein secretory vesicles (Fig. 3E). It still remains to be determined whether calpactin plays a merely structural role in lactating marnniary cells or whether it is involved in constitutive and/or regulated exocytosis. It is intriguing that in secreting mammary cells the casein-containing secretory vesicles were found to be linked to the plasma membrane before fusion by short cross-links reminiscent of those formed by calpactin bound to
Cytoskeleton: its Role in Cellular Function
Fig. 3 lmmunoperoxidase localization of calpactin (annexin II) in lactating mouse mammary gland Vibratome sections of 10 day, post-partum mouse mammary gland were stained with anti-cal ( I I5), processed using the immunoperoxidase technique and prepared for electron microscopy [25] (A) Dark reaction product is present along the apical membranes but, not the basal (arrowheads) membranes of mammary epithelial cells (B) Unstained apical region of cell (C) Stained apical region of mammary cell showing plasma membrane and microvillar staining Secretory vesicle membranes and sites of compound exocytosis were also stained by the antiserum (arrows in D and E) Scale bars, I p m
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lipid vesicles and observed in stimulated chromaffin cells [U]. The results summarized here have allowed us to begin to compare exocytotic secretion in two very different cell types. Continued characterization of adrenal chromaffin and mammary cells should give further insight into the similarities and differences between the regulated and constitutive exocytotic pathways.
0 . Hurgoyne, K. I)., (;eisow. 51. J. 81 Harron, J. (1982) I’roc. K. Soc. Idondon H 216, 1 1 1-1 15 7. Cheek, 7’. K. & Ihrgoyne. K.L). (1991) The Neuronal Cytoskeleton (Ihirgoync. I
