All eukaryotic cells, including the pancreatic acinar cell, tightly control organization of their cytoplasm which in large part relies on the cytoskeleton. The cytoskeleton is comprised of three filament types: microtubules (tubulin), intermediate filaments (keratins), and microfilaments (actin). In addition to characteristics common to all cells, the pancreatic acinar cell is a polarized epithelial cell with a vectorial arrangement of the secretory machinery and apically-directed protein secretion, with important roles of the cytoskeleton in this polarity. The most investigated of the cytoskeleton elements in the acinar cell are actin-based microfilaments which are involved in regulated protein secretion. The development of the fluorescent Lifeact probe for realtime visualization of actin dynamics in live cells makes this an exciting time for the field. The dynamic rearrangement of the actin cytoskeleton associated with protein secretion is the focus of this review.
The actin cytoskeleton that is ubiquitous in nonmuscle cells is largely made up of b-actin and g-actin, the products of the ACTB and ACTG1 genes, respectively (2). These two actins differ by only 4 amino acids but they appear to have different functions, as mutations in the two genes have very different effects (2). In the mouse acinar cell, Actb mRNA is expressed more than 2-fold greater than Actg1 (Table 1); presumably their protein levels match this expression although this has not been measured in the pancreas. In gastric parietal cells, b-actin is more localized to the apical cortical cytoskeleton whereas g-actin is more localized along the basolateral plasma membrane (BLPM) (48). If the same is true in the acinar cell, then b-actin is more likely to be important in protein secretion which is also localized to the apical surface of the cell. In the acinar cell, g-actin may be involved in the BLPM blebs that form under supraphysiologic stimulation. From here on, actin will be referred to as if it were a single gene/protein.
Results from studies of the actin cytoskeleton in the exocrine pancreas are the primary source for this review. However, there are many gaps in our knowledge regarding exocrine pancreas function. When data from pancreas are lacking, results from other cell types that can reasonably be assumed to be true of the acinar cell are briefly discussed. It should be noted that even though there are commonalities shared by all cells with a regulated secretory pathway [e.g., regulation by small GTPases and use of SNAREs to mediate granule/vesicle - plasma membrane (PM) recognition and fusion (19)], there can be important differences.
For example, the role of actin coating of granules/vesicles during the content release process may serve different functions related to the speed of product release required (see 4 E. Dynamics of acinar actin in the stimulated state below).
Actin is a 42 kDa protein that exists in two distinct forms in the cell: monomeric (globular or G-actin) ; and polymeric (filamentous or F-actin). Actin monomers have the inherent behavior of self-assembly into filaments and prevention of inappropriate polymerization is controlled by several G-actin binding proteins that stoichiometrically outnumber G-actin by more than 3-fold (47). However, in general the majority of a cell's actin is in the form of F-actin, estimated to comprise ~75% of the total (47). The structure of F-actin microfilaments (MF) is a polarized double helix such that the barbed (+) end is where new monomers are primarily added and the pointed (-) end is where F-actin primarily releases actin monomers. Actin is an ATPase, and for G-actin the ATP-bound form predominates and is thus primed for addition to an actin filament. Once added to the filament, the ATPase is activated and ATP is hydrolyzed to ADP.
F-actin is highly dynamic and there are numerous proteins that regulate the assembly and disassembly of MF. Interrogation of a published microarray study done in the author's lab(18)for actin cytoskeleton elements was performed and is presented in Table 1. This yielded a number of genes, the most highly expressed of which not surprisingly were b- and g-actins, as well as several proteins known to bind to or regulate dynamics of actin. Many of these proteins directly interact with actin and are thus called actin binding proteins (ABP) (42). The majority of genes on this list and their functions were discovered using other cell systems and have not been investigated in the pancreatic acinar cell. Microfilament (MF) structures in cells may appear to be highly stable structures, but they are constantly undergoing growth at the (+) end and disassembly at the (-) end, a process called treadmilling (38). Thus, the apparent stability of MFs is actually due to a dynamic equilibrium. It has been estimated that the ~1 mm long MFs of the intestinal microvillus turns over every 20 min by treadmilling, and a similar rate is also believed to be true of the terminal web (TW) that underlies the apical plasma membrane (APM) (9). In addition to proteins that induce disassembly of MFs, there are numerous actin-binding proteins that regulate nucleation of new fibers and growth of existing fibers. Actin can exist as higher order bundles of multiple MFs in linear arrays as well as branched MF structures, with accessory proteins providing lateral cross-links (42).
Actin Localization and Dynamics in the Acinar Cell
Methods to Visualize F-actin
Most studies of actin in the acinar cell have been of MFs and usually after chemical fixation of the cells in either the unstimulated basal state, with a secretory stimulus, and/or after a specific experimental manipulation of the cells. The most commonly used technique is fluorescently tagged phalloidin, a fungal toxin with a high affinity for F-actin (see Pancreapedia articles and images therein: Rab3; Visualization of exocytosis in pancreatic acinar cells by fluorescence microscopy; Rab27). An example of FITC-phalloidin labeling of mouse acini from the author's lab is shown in Figure 1, which shows the MFs of acinar cells under basal and stimulated conditions. Phalloidin is a stabilizer of F-actin MFs so when used in living cells it perturbs the actin cytoskeleton. This property limits use of phalloidin to static imaging of F-actin after fixation, and precludes its use to visualize dynamics of actin in live cells where other techniques are used (see below).
F-actin can also be recognized in standard transmission electron microscopy as electron dense ~6 nm diameter filaments (smaller than microtubules, ~24 nm; and intermediate filaments, ~10 nm).
Until recently, MFs have been difficult to visualize in living cells without perturbing the structure and function of the cytoskeleton. Adding tags such as green fluorescent protein (GFP) to b-actin perturbs the actin cytoskeleton into which it is incorporated [discussed in (14)]. A newer approach is to make chimeric fluorescent proteins from ABPs. Using such an approach the Lifeact reporter was made, which consists of a 17 amino acid actin binding domain from the ABP140 protein, fused to GFP (31). This chimeric protein has a 30-fold greater affinity for F-actin than G-actin, was found to not interfere with known activities of the actin cytoskeleton, and it faithfully reproduces known F-actin staining patterns in a variety of cell types even under dynamic situations where the actin cytoskeleton is being remodeled (14). This construct was later used to make a transgenic mouse expressing the fluorescent F-actin reporter in all tissues, called the Lifeact mouse (32).
G-actin can also be visualized using fluorescently tagged DNase I, which binds to both G- and F-actin, with higher affinity to the former (21), although an analysis of DNAse I inhibition by highly purified G- and F-actin concluded that both forms have similar affinity for DNase I (25). An example of fluorescent DNase I staining of G-actin in the acinar cell shows a diffuse cytoplasmic distribution which is not very informative (27). Visualization of G-actin has not been used to study the actin cytoskeleton of the acinar cell to any extent. Nor has there been systematic investigation of G-actin ABPs in the acinar cell.
Acinar Cell Actin in the Resting State
The single largest pool of F-actin in the acinar cell is in the TW (4), also called subapical actin or cortical actin (Figure.1, left panel).
The TW is a dense arrangement of MF that forms a continuous sheet-like network ~200 nm thick (13) under the APM. Among ABPs, members of the red blood cell spectrin family cross-link TW MFs and also link the TW to perpendicular MF that extend into the microvilli (MV), as shown in intestinal epithelium (16). As compared to the villus epithelium of the small intestine where MV are best characterized, the acinar cell MV are relatively sparse. A second actin-based structure is a ring of MFs that are attached to the adherens junctions near the apical surface and encircle each acinar cell. Because this is a ring-shaped structure it is less easily visualized than the sheet-like TW. There are also recently-described MF bundles that project perpendicularly from the TW into the cytoplasm that are called actin cables (15). Lesser amounts of MFs are found along the BLPM of the acinar cell (Figure 2, left panel). Because of the large amount of F-actin in the TW, it can be difficult to visualize the other sites of MFs when using fluorescent labeling approaches. For example, the recently described actin cables that run perpendicular to the TW in acinar cells are not observed using phalloidin staining, but are visualized using a chimeric ABP domain fused to GFP (Lifeact, see below) (15).
Effects on Secretion of Pharmacological Agents and Molecular Approaches that Perturb the Actin Cytoskeleton
There is a large data set about the acinar cell actin cytoskeleton obtained using a variety of approaches. Because the actin cytoskeleton is so important to eukaryotic cells, there are numerous naturally occurring substances produced by a variety of organisms that interfere with the organization of actin, and many of these are available as research tools. Also, investigators have produced constitutively active (CA) and dominant negative (DN) constructs to stimulate or inhibit upstream pathways that control the actin cytoskeleton. A summary of these toxins and reagents and their effects on the actin cytoskeleton and acinar cell amylase release is given in Table 2. Some of the reported effects of a particular agent are contradictory but these are likely attributable to differences in concentration of the agent (e.g. latrunculins have been used over the wide range of 0.3 mM to 100 mM), time of exposure, and temperature at which the experiment was performed [discussed in (7)]. The importance of a specific agent's concentration is illustrated by the finding discussed below that a small perturbation of the actin cytoskeleton increases amylase release, whereas stronger perturbation blocks amylase release (26). The data covered here attempts to give the consensus of the available data.
Signals that Affect the Actin Cytoskeleton
The principal mechanism of acinar cell stimulation begins with ligand binding to membrane receptors coupled to G proteins that feed into several pathways. With respect to the acinar cell actin cytoskeleton, the known pathways are activation of the small GTPases RhoA (via Ga13) and Rac1 (via Ga13 and Gaq) (35). These activated small GTPases in turn activate their downstream effectors such as Diap1 and ROCK1; and WASP/WAVE and ARP2/3, respectively, that lead to actin cytoskeleton remodeling.
As discussed below, a major downstream effector of Rho activation is the formin family member Dia. In drosophila, the formin Diaphanous is activated by the small GTPase Rho, resulting in nucleation and formation of linear F-actin filaments that comprise the TW of epithelial cells (23). Although not yet experimentally verified, Rho activation in the mammalian pancreas is expected to activate mDia1 and it has been shown in mouse acinar cells that constitutively active (CA)-mDia1 leads to F-actin cables perpendicular to the apical PM (15). It was suggested these F-actin cables serve to recruit zymogen granules (ZG) through the TW to the apical membrane during secretory stimulation (15). Consistent with the requirement for apical MF in secretion, protein release into the tracheal lumen of Drosophila lacking Dia, which also lack TW F-actin, was absent. A notable difference between Drosophila and mammalian acinar cells is that Dia in drosophila is required for formation of the TW of epithelia (23); whereas in mouse acini, the only observed role of mDia was in formation of the F-actin cables perpendicular to the APM, and the TW persisted in the presence of a DN-mDia construct (15). Also in contrast to Drosophila, mouse acini expressing dominant negative (DN)-mDia1 have unchanged basal and stimulated amylase release (15). Clearly, the roles of Dia/mDia1 differ in Drosophila and mouse pancreatic acini.
Dynamics of Acinar Actin in the Stimulated State
Actin undergoes a variety of changes during secretory stimulation. The TW in the resting acinar cell is a barrier to zymogen granule (ZG) exocytosis, and the F-actin here must be rearranged to allow ZG access to the APM when the cell is stimulated (26). Using streptolysin O-permeabilized acinar cells, it was demonstrated that mild perturbation of the actin cytoskeleton by adding low concentrations of G-actin sequestering proteins (b-thymosin or a gelsolin fragment; Table 2) resulted in amylase release, especially the early phase of release (26). Interestingly, even when Ca2+ was chelated at low levels using EGTA, mild perturbation of the actin cytoskeleton resulted in amylase release. If high concentrations of the G-actin ABPs were used that resulted in extensive loss of F-actin, there was reduced amylase release. Similarly, if the F-actin stabilizing drug phalloidin was introduced to permeabilized acini, amylase release was inhibited. Together, these data indicate that the TW is a barrier to ZG exocytosis and that, while some remodeling of the actin cytoskeleton is required for release, extensive disruption or stabilization of F-actin is detrimental to protein secretion.
The dynamics of G-actin per se have not been investigated in relation to acinar cell stimulation. However, there have been measurements of the relative changes in the total amount of F-actin in acinar cells, which show that an increase in polymerized actin is associated with amylase release (6). Furthermore, pharmacological reduction of F-actin (1.0 mM latrunculin, a G-actin sequestering agent, Table 2) inhibits CCK-stimulated amylase release from mouse acini without affecting Ca2+ dynamics (7). Since an acute increase in F-actin means less G-actin, these data add to the concept that dynamic changes in actin are involved in protein secretion in the acinar cell.
A similar conclusion regarding actin remodeling during secretion can be drawn from experiments using supramaximal CCK which induces large alterations in the actin cytoskeleton and shows inhibition of amylase release (10, 40). When DN-Rho and DN-Rac (Table 2) constructs were used, they prevented supramaximal CCK inhibition of amylase release, and at the same time lessened reorganization of the actin cytoskeleton (6). In that study a low dose of jasplakinolide (1 mM) also prevented supramaximal CCK inhibition of amylase release, but a higher dose (3 mM) inhibited amylase release at all concentrations of CCK, consistent with the effect of jasplakinolide reported by Valentijn et al (43) (Table 2). So again, these results show there is a 'sweet-spot' for amylase release with respect to organization of the acinar cell actin cytoskeleton.
Remarkably, during exocytosis the ZG membrane (ZGM) becomes 'coated' with F-actin (actin-coated granules, ACG) (43) (Figure 1, right panel). In the acinar cell, the role of the F-actin coat has been proposed to stabilize the fused ZG at the PM (20, 41). This may be related to the duration of acinar protein secretion (seconds, which is long relative to neuronal exocytosis which occurs on a millisecond time scale), which may be required for the highly concentrated digestive enzymes to solubilize and diffuse out of the granule lumen into the beginning of the pancreatic ductal system. In other cells with the regulated pathway whose contents are more rapidly solubilized, the F-actin coat appears to flatten the fused vesicle thus quickly forcing out the secretory product. The potential differences in the roles of F-actin granule/vesicle coats in various secretory cells are discussed in some excellent reviews (28, 29). Interestingly, the G-actin sequestering drug latrunculin blocks coating of ZG with actin but does not prevent ZGM-PM fusion (17).
Using the C3 Rho toxin from Clostridium botulinum, it was shown that ACG formation is Rho-dependent (27). When acini were pretreated with the C3 toxin and then stimulated to secrete, ACG were not observed but large vacuoles formed that were filled with the fluorescent extracellular tracer, showing that granule fusion had still occurred but that the individual granules in the absence of actin coating appear to fuse into a large vacuole. Thus, the rapid formation of ACG in stimulated cells appears to be Rho-dependent, and ACG formation may prevent vacuole formation. Amylase release was not measured in these experiments.
The timing of F-actin coating of ZG relative to ZGM-PM fusion has evolved since its discovery. Early descriptions of ACG suggested that the coat developed before ZGM-PM fusion, based on failure of entry of a fluorescent lectin (WGA) from the medium into ACG, indicating their lumens were not yet accessible to the medium (43). Later experiments concluded that the F-actin coat forms after membrane fusion, while the exocytic pore is still open (27, 41) (also see Pancreapedia article Visualization of exocytosis in pancreatic acinar cells by fluorescence microscopy). This conclusion was based on the observation that only ZG accessible to the acinar lumen (able to take up lysine-fixable fluorescent dextran from the medium) were actin-coated and no ZG without fluorescent dextran were actin-coated. An example of acini stimulated in the presence of a lysine-fixable dextran followed by fixation and fluorescent phalloidin staining is shown in Figure 2. This clearly shows dextran in the lumens of ACG. Subsequent work using a soluble tracer (sulforhodamine B) with living cells convincingly showed that access to the ZG lumen occurred on average 6.7 sec before appearance of the actin coat, as visualized in acini from a transgenic mouse expressing Lifeact-GFP (17) (Figure 3). This study also showed that latrunculin blocked formation of ACG while ZG fusion was not inhibited (Figure 4). Hence, ZGM-PM fusion occurs rapidly followed by actin coating of the fused ZG while it is still in the 'Ω' shape, and that fusion per se is independent of ACG formation.
A different interpretation of the timing of ZG fusion and ACG formation has been made recently by Geron et al., using Lifeact-GFP, expressed in acini by adenoviral infection (15).
This study was of the role of the formin family member diaphanous (DIAP1, called mDia in mouse), which is a Rho effector whose activation leads to linear MFs in the pancreatic acinar cell that they called actin cables. They state in the introduction to their paper that 'Secretory vesicles undergo actin coating before fusing to the apical membrane...' (emphasis added) and they cite (27, 28, 43). One can observe in their videos accompanying the paper ACG moving apparently along F-actin cables that run perpendicular to the apical PM. Since these ACG appear deeper inside the cells than the PM, it was concluded that coating preceded ZGM-PM fusion. Even though they included FM4-64, a fluorescent membrane tag that should enter fused ZG and label their membranes, in none of the data shown is FM4-64 fluorescence ever observed in ZG. Therefore, it is not clear that the experiments actually could reveal when ZGM-PM fusion occurred. A more consistent interpretation of the role of mDia-induced actin cables is that fused ZG are moving along these cables near the apical PM.
Another issue with their interpretation is that it does not concur with what was observed when the cables were interfered with. They showed that expression of a CA- mDia1 resulted in an increase in actin cables, and that a DN-mDia1 mutant caused a strong decrease in the number of cables. Neither of these affected basal or stimulated amylase release. If the cables are an important mechanism to move ZG in the cytoplasm to the PM so they can then fuse, one would expect a decrease in stimulated amylase release when the cables are decreased, which was not observed. The DN-mDia1, which reduced cable formation, caused an increase in ACG fused to one another, which was interpreted as compound exocytosis. So, it would appear that the role of these mDia1 induced actin cables is to keep ZG separated such that individual granule fusion predominates and minimizes compound exocytosis.
It is likely an issue in these disparate results regarding the timing of ACG formation whether the acinar preparation was optimal for allowing access from the medium to the acinar lumen. This is discussed in (20, 46), where it is shown that the vigor of the tissue dissociation technique can yield groups of cells ranging from multi-acinar clusters to single acini or even smaller than acinar-sized groups that retain normal acinar secretory function. To allow access of the bathing solution to the acinar lumen, groups consisting of as few as 3-6 cells with a short unhindered connection between the acinar lumen and the medium is optimal. [In my lab, we could not get lysine-fixable dextran into fused ZG (37) as achieved by other labs, likely because my 'acini' were actually larger multi-acini clusters.]
Although mDia is a downstream effector of activated Rho, introduction of DN-mDia (15) and DN-Rho A (6) in acinar cells does not produce the same effects (Table 2). DN-mDia does not affect amylase release whereas DN-Rho inhibits amylase release. These different effects on amylase release indicate that mDia is not the sole target of activated Rho in the acinar cell, and perhaps another of the Rho effectors is involved (39).
The conclusion that the F-actin coat develops rapidly after ZGM-PM fusion contributes to the idea that this coat functions to stabilize the pore in the open state, providing sufficient time for the stored zymogens to become soluble and diffuse into the acinar lumen. An actin coat attached along the cytosolic surface of the fused ZG would stiffen this membrane, preventing closure of the energetically-unfavorable pore. The presence of the nonclassical myosin Vc (Myo5c) on ZG, possibly tethered to the membrane by the small GTPase Rab27B (11, 12) is believed to provide a motive force to move ZG along the TW. Another myosin, Myo2A, is localized to the apical pole of acinar cells, is phosphorylated when the cell is stimulated, and when its activity is inhibited access of the ZG lumen to the soluble markers in the medium is reduced (5). This was interpreted as more rapid fusion pore closure when this myosin activity is inhibited. Also, when the TW was disassembled using cytochalasin treatment, the acinar lumen enlarged under stimulation but amylase was not released; when the cytochalasin and secretory stimulus was washed out, the TW recovered and amylase was released from the already fused ZG (44). It was suggested that contractile forces on the fused ZGM by actin were needed to force the ZG content out. These results can be reinterpreted in light of the newer data indicating that MFs are required to maintain an open pore for protein release.
In addition to providing sufficient time for zymogens to dissociate and diffuse into the lumen, the actin-coat of fused ZG has been proposed to help counteract the expected hydrostatic pressure of these dissolving contents from the fused ZG in salivary glands (22). Also, there is compelling evidence that the fused ZGM transports electrolytes and fluid from the acinar cytosol into the lumen during protein secretion (see review in the Pancreapedia by Frank Thévenod; Channels and Transporters in Zymogen Granule Membranes and their Role in Granule Function: Recent Progress and a Critical Assessment) which will add to hydrostatic pressure within the fused ZG lumen.
While it has been shown that mDia1 is involved in the formation of actin cables in the acinar cell, and is regulated by the small GTPase Rho, it remains to be determined what controls localized disassembly of the TW required to allow passage of ZG to the PM for exocytosis. The F-actin structure that coats granules is likely to consist of a network of short interconnected linear filaments, or alternatively, of branching actin filaments. This is because linear F-actin filaments <10 mm in length are rigid rods (8) and such long linear filaments could not be accommodated on the highly curved surface of granules which are ~1 mm in diameter (34). Consistent with this idea, a subunit of the ARP2/3 complex and N-WASP, factors involved in nucleation of branches on existing MFs, were immunolocalized to the region of ACG in stimulated acini (15). Based on this, Rac1 may be involved, because Rac1 has been shown to regulate WASP/WAVE ABPs that control nucleation/elongation of branched MF in other systems (30). However, WASP nucleates a new branch on existing F-actin and proteomic analysis of highly purified ZGM revealed the presence of small GTPases and molecular motors (dynein, myosins) but not actin or other cytoskeletal elements (11, 12, 33). On the other hand, ultrastructural immunogold localization showed actin in the region of ZG (1, 3). Together these results suggest that actin in the ZG region of the cell is either loosely bound or not bound at all to the ZGM. In any case, it appears F-actin is not present on ZG before fusion, so there needs to be a process to nucleate new MF on the fused ZGM and at this time, how this occurs remains to be identified.
After the Stimulus Ends
Once the fused ZG has released its content and exocytosis can be considered complete, the actin cytoskeleton has the further role of helping drive compensatory endocytosis. In order to maintain the balance of membranes between apical and basolateral domains, the ZGM added to the apical surface during protein secretion must be taken back into the cell. The endocytic process is believed to retrieve the ZGM in a clathrin-mediated fashion, and this also requires the actin cytoskeleton (44). It was observed in cytochalasin D treated acinar cells that the APM became dilated and pits on this surface were immunoreactive for the clathrin adaptor AP-2, clathrin, dynamin, and caveolin, all of which are components of various endocytic mechanisms. When cytochalasin was washed out, these proteins rapidly disappeared from the APM and the luminal membrane recovered to its prestimulus size. From this it was suggested that the actin cytoskeleton is also involved in the compensatory endocytosis process that follows protein secretion. The roles of actin in endocytosis are well known (24).
There are several filamentous actin structures with important roles in acinar cell protein secretion: the TW, ACG, and actin cables. There is also BLPM associated F-actin which is involved in membrane blebbing under supramaximal stimulation. The recent development of the fluorescent Lifeact probe has begun to reveal the dynamics of the actin cytoskeleton with new insights into post-fusion actin coating of ZGs and the discovery of actin cables perpendicular to the TW. It is expected this tool will continue to be utilized for greater in depth understanding of the actin cytoskeleton and its roles in protein secretion from the acinar cell.
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