actin dynamics are central to a number of basic physiologies in polarized cells, including polarized cell domains, cell shape, and intracellular trafficking and signaling. The study by Ammar and colleagues, the current article in focus (Ref.1, see p. C407 in this issue), provides evidence in gastric parietal cells for the regulation of actin pools of varying stability and turnover. The investigators have utilized latrunculins that sequester G-actin monomers, thereby reducing the pool of polymerizable actin. This mechanism is substantially different from cytochalasin D, which leads to fragmentation of F-actin filaments. Thus latrunculin is not expected to alter stable F-actin structures. However, in situations where there is turnover of F-actin, latrunculin treatment can deplete cellular F-actin. In support of this concept, these investigators have observed little effect of latrunculin B treatment on resting parietal cells. In contrast, significant effects were observed in cells stimulated with increases in cAMP. In cultured parietal cells, the investigators were able to follow two separate cAMP-stimulated actin-based functions that were associated with actin turnover: lamellapodial spreading along the Matrigel substratum and vesicle fusion into canalicular vacuoles that are lined by F-actin. Although both functions could be altered by treatment with latrunculin B, alteration of canalicular F-actin and inhibition of aminopyrine accumulation (an indirect assay of tubulovesicle fusion) required at least 10-fold higher concentrations of latrunculin than that which is needed for inhibition of lamellapodial spreading. These results suggest the presence of distinct pools of basolateral and apical F-actin filaments in parietal cells.
The presence of separable pools of actin in polarized epithelial cells underscores the subcellular specializations required for a physiological response to global signals such as increases in intracellular cAMP. Yao and colleagues (11) have previously demonstrated that the apical canalicular membrane in parietal cells is underlined by F-actin filaments that contain β-actin, while the basolateral membrane contains γ-actin. Separation of these two pools of F-actin containing different actin isoforms accounts for the polarized distribution of actin-binding proteins such as ezrin, which is predominantly associated with the apical membranes through its binding with β-actin filaments (11). It is of note that evidence does exist for movement of proteins between these two pools of F-actin in parietal cells. Chew and colleagues (5) have noted in parietal cells the redistribution of Lasp-1, a multidomain linker protein, from basolateral γ-actin filaments to the secretory canaliculus in response to increases in intracellular cAMP. The effects of latrunculins observed in the present report could therefore reflect either differential binding of latrunculins to different γ-actin monomer isoforms of actin or differences in the rates of F-actin turnover.
The parietal cell recycling of the H+-K+-ATPase remains the most dramatic example of apical membrane recycling (7). Although this process is massively amplified in parietal cells, it is a fundamental component of polarized cell function in all epithelial cell systems. Recent studies have demonstrated that many of the candidate regulators found on parietal cell tubulovesicles, including Rab11 small GTPases, soluble N-ethylmaleimide-sensitive factor attachment protein target receptor proteins (SNAREs), and secretory carrier membrane proteins (SCAMPs), are also present on vesicles associated with general apical recycling systems in cultured cell systems such as Madin-Darby canine kidney (MDCK) cells (2-4, 6, 10). However, in contrast with parietal cells, investigations of general apical recycling systems in cultured cell line systems have emphasized the important role of microtubules in the coordinated processes of apical recycling and basolateral-to-apical transcytosis (2). Disruption of microtubules in these systems markedly inhibits both transcytosis and apical recycling. In contrast, the present investigations, as well as previous studies in parietal cells (11), all emphasize a prominent role of the actin-based cytoskeleton in the apical recycling of the H+-K+-ATPase. Microtubule disruption appears to a have a relatively minor effect on parietal cell function.
How can one rationalize these disparate results in different cell systems? First, recent investigations have demonstrated that an actin-based motor, myosin Vb, associates with Rab11a and is required for cargo exit from the apical recycling system in MDCK cells (9). Thus the actin-based cytoskeleton may be a general regulator of movement of recycled cargoes out from apical recycling systems toward eventual fusion with apical membrane targets. Second, it is possible that the parietal cell, in addition to amplifying the apical recycling system, has also specialized specifically in this process to the exclusion of other vesicle trafficking functions possessed by most polarized epithelial cells. Many model systems, such as MDCK cells, utilize the apical recycling system for processing of both apically recycling and transcytosing cargoes, with the transcytotic pathway accounting for the majority of the membrane trafficked (2). In contrast, the parietal cell likely processes little if any transcytotic traffic. Thus the microtubule-based dependence of the apical recycling system in MDCK cells may reflect the requirement for microtubules for the entry of the predominant source of transcytosing membranes into the system. No studies have examined the effects of latrunculins on apical recycling or transcytosis in MDCK cells. Still, it is important to note that the trafficking of another apically recycled protein, Na+/H+ exchanger 3, is altered by latrunculin treatment (8). The absence of microtubule dependence in parietal cell recycling may reflect its purely actin-based recycling pathways without any need for membrane pools that require microtubules for entry into the recycling system.
The studies presented in the present report establish the polarization of separate actin pools with differentiable functions at the basolateral and apical membranes. Future clarification of the requirements for actin-based vesicle movements out from apical recycling systems in cell lines such as MDCK should provide an increased understanding of the general role of the actin cytoskeleton in regulating membrane recycling. Similarly, further investigations must now address how the elements of the actin cytoskeleton interact with tubulovesicle proteins to mediate and modulate vectorial fusion of H+-K+-ATPase-containing vesicles with the parietal cell secretory canaliculus.
Address for reprint requests and other correspondence: J. R. Goldenring, Institute of Molecular Medicine and Genetics, CB-2803, Medical College of Georgia, 1120 Fifteenth St., Augusta, GA 30912 (E-mail:).
- Copyright © 2001 the American Physiological Society