Panx1 regulates neural stem and progenitor cell behaviours associated with cytoskeletal dynamics and interacts with multiple cytoskeletal elements
© Wicki-Stordeur and Swayne; licensee BioMed Central Ltd. 2013
Received: 15 July 2013
Accepted: 19 August 2013
Published: 21 August 2013
Pannexins (Panxs) are relatively newly discovered large-pore ion and metabolite permeable channels. Although no proteomics-based interactome has yet been published, Panx1 has been demonstrated to interact with actin in an ectopic expression system. This interaction affects both Panx1 plasma membrane stability as well as cytoskeletal remodelling. The current study builds on our recent discovery of Panx1 expression in ventricular zone (VZ) neural stem and progenitor cells (NSC/NPCs), and on the demonstrated interaction of Panx1 with the cytoskeleton.
Here we demonstrate that Panx1 also plays roles in two additional cell behaviours associated with neurogenesis, including cell migration and neurite extension. Furthermore, we confirm an endogenous interaction between actin and Panx1, and identify a new interaction with actin-related protein 3, an actin cytoskeleton-modulating protein.
This study further establishes the importance of Panx1 in the cell biology of NSC/NPCs and strengthens and expands our knowledge of Panx1 interactions with the cytoskeleton.
Panxs are four-pass transmembrane proteins that oligomerize to form large-pore mechanosensitive channels permeable to ions and metabolites of up to 1 kDa in size, such as adenosine triphosphate (ATP) [1, 2]. We recently detected Panx1 expression in the Neuro-2a (N2a) cell line as well as in post-natal ventricular zone (VZ) neural stem and progenitor cells (NSC/NPCs), where it positively regulates cell proliferation in part through release of ATP that results in activation of purinergic receptors. This built on earlier work demonstrating an important role of constitutively released episodic bursts of ATP in the proliferation of VZ NSC/NPCs, which in turn activates metabotropic purinergic receptors in an autocrine and paracrine manner [3–5]. Perhaps not surprisingly, as a mechanosensitive channel, recent work has shown that Panx1 is actually physically linked to the cytoskeleton. In an ectopic expression system, Panx1 was reported to physically interact directly with actin . A recent study in glioma cells further supported a role for Panx1 in the dynamic regulation of actin cytoskeleton remodeling . Here we extend on our previous discovery of Panx1 expression in VZ NSC/NPCs by further defining the cell-type demographics of Panx1 over the course of VZ neurogenesis, by demonstrating that Panx1 plays a role in additional cell behaviours associated with neurogenesis, including cell migration and neurite outgrowth, and by uncovering additional interactions with cytoskeletal elements, further establishing the relationship of Panx1 with the cytoskeleton.
Dcx-positive neuroblasts overall appeared to express less Panx1 than NSC/NPCs immediately proximal to the ventricle, suggesting that Panx1 expression decreases with differentiation. This was confirmed in vitro in retinoic acid and low serum differentiated N2a cells and differentiating VZ NSC/NPC cultures (Figure 3A,B). Interestingly, immunostaining of endogenous Panx1 in neuronally differentiated VZ cells and N2a cells revealed strong Panx1 expression within the developing neurites (Figure 3A,B). Since differentiation of N2a cells and VZ NSC/NPCs in vitro is associated with such marked neurite outgrowth, we examined whether blocking or knocking down Panx1 can, on its own, induce neurite extension in the absence of additional differentiation stimuli. Indeed, blocking Panx1 with probenecid [12–14] induced marked neurite extension in both N2a cells and VZ NSC/NPCs (Figure 3C-I). Similarly, Panx1 siRNA knockdown in N2a cells caused increased neurite outgrowth without additional stimuli (Figure 3J), while Panx1EGFP overexpression inhibited neurite extension in N2a cells induced to differentiate (Figure 3K).
We discovered several novel interactions, including several cytoskeleton-related proteins. Gene ontology (GO) analysis (http://www.broadinstitute.org/gsea/msigdb/index.jsp) of hits revealed that 10% of the putative Panx1-interacting proteins amenable to GO analysis could be classified by the generic GO term GO:0005856 aka ‘CYTOSKELETON’ (Additional file 2: Table S1). This GO term refers to ‘Any of the various filamentous elements that form the internal framework of cells, and typically remain after treatment of the cells with mild detergent to remove membrane constituents and soluble components of the cytoplasm. The term embraces intermediate filaments, microfilaments, microtubules, the microtrabecular lattice, and other structures characterized by a polymeric filamentous nature and long-range order within the cell. The various elements of the cytoskeleton not only serve in the maintenance of cellular shape but also have roles in other cellular functions, including cellular movement, cell division, endocytosis, and movement of organelles.’
A large number of these newly identified cytoskeleton-associated proteins (14/26) were associated with the GO term collectively known as ‘ACTIN_CYTOSKELETON’ (GO:0015629). Among these, we observed significant overlap of actin-related protein 3 (Arp3; Figure 4B,C) and actin (Figure 4D) with Panx1EGFP by confocal microscopy. Interestingly, as with endogenous Panx1, we also observed high levels of Panx1EGFP localized to neurites and other cellular protrusions resembling filopodia (Figure 4C,D). Finally, endogenous Panx1, actin and Arp3 co-precipitated from N2a cells (Figure 4E). Actin was previously identified as a Panx1 interactor in cells ectopically expressing Panx1 ; this confirms that an interaction occurs between the two endogenously expressed proteins.
The novel Panx1 interactor, Arp3, is a major component of the Arp2/3 complex, a seven-subunit protein that plays a major role in the regulation of the actin cytoskeleton (reviewed in Firat-Karalar and Welch, 2011 ). A link between Panx1 and actin cytoskeleton rearrangements has previously been described . Arp3 closely resemble the structure of monomeric actin, and one of its functions is to serve as a nucleation site for new actin filaments. Actin and actin-associated proteins, including the Arp2/3 complex, have been shown to be integral in both migration [18, 19] and neurite outgrowth [19, 20]. Actin polymerization in lamellipodia and filopodia of migrating cells provides the necessary driving force for leading edge protrusion. Furthermore, Arp2/3 complex regulates the actin filaments present in these cellular processes. In fact, Arp2/3 depletion has been shown to significantly reduce filopodia formation in both primary neurons and neuroblastoma cells , while alterations to Arp2/3 function cause dysregulation of lamellipodia dynamics . Furthermore, Arp2/3-mediated actin polymerization regulates growth cone mobility and neuritogenesis , as loss of Arp2/3 activity causes erratic neurite numbers and extension, as well as increased focal adhesions . Moreover, in agreement with our previously published work illustrating a role for Panx1 in the positive regulation of VZ NSC/NPC proliferation, the actin cytoskeleton has been shown to be indispensable for cell division (reviewed in Firat-Karalar and Welch, 2011 ). This includes roles in contractile ring formation, centrosome separation, and spindle positioning. As Panx1 interactors, actin and Arp3 have illustrated a direct connection between Panx1 and the actin cytoskeleton. This further supports the observed role for Panx1 in the actin-associated behaviours of cell migration and neurite outgrowth, as well as that previously published linking Panx1 to cell proliferation.
Altogether, our data expand on our previous findings by demonstrating that Panx1 is expressed in Dcx-positive migrating neuroblasts in adult brain, and is also involved in additional cell behaviours associated with neurogenesis, including migration and neurite outgrowth. Further, our analysis of protein interactions uncovered a novel Panx1 interacting protein, Arp3, a major part of the Arp2/3 complex, which is an important regulator of actin cytoskeletal dynamics in cell proliferation, neuritogenesis and cell migration . We also established that endogenously expressed Panx1 interacts with actin, and discovered that a large proportion of Panx1 interacting proteins are associated with the cytoskeleton. Overall, this study provides novel evidence reinforcing the link between Panx1 and the cytoskeleton, and suggests that this relationship underlies the regulation and function of Panx1 in VZ NSC/NPCs.
Operating support for this work came from a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant, a Victoria Foundation Willard and Elva Dawson fund grant, and a University of Victoria laboratory start-up grant awarded to LAS. We also thank the Canadian Foundation for Innovation Leaders Opportunity Fund, and the British Columbia Knowledge and Development Fund for supporting the purchase of a confocal microscope that was essential to this work. LWS is supported by an NSERC Vanier Canada Graduate Scholarship, a Howard E. Petch Research Scholarship and an Edythe Hembroff-Schleicher Graduate Scholarship. Finally, we thank Ross Prager for assistance with data analysis, and Jen Graham for assistance with animal work.
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