Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca²⁺-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP₃ and Ca²⁺ that initiate the propagation of the Ca²⁺-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca²⁺-wave propagation are provided by gap junction channels through the direct transfer of IP₃ and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca²⁺-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca²⁺-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 μm. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca²⁺-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.     

CARNOY: A new digital measurement tool for palynology

Quantitative data play an important role in palynological research. With the advent of digital imaging in light and electron microscopy, palynologists now have the opportunity to perform measurements faster and more precisely than ever before. Several image analysis software packages already exist for these tasks, but they are often expensive, difficult to use or not adapted to the specific needs of palynologists. After studying the daily workflow of a palynologist, we designed CARNOY, an image analysis application written from the ground up for use in palynology and morphology. CARNOY offers an easy-to-use interface and several features to make measuring easier and faster. The program can export measurements to almost every other software package for further analysis and is available for free on the Internet.     

Mapping functional interactions in a heterodimeric phospholipid pump

Type 4 P-type ATPases (P(4)-ATPases) catalyze phospholipid transport to generate phospholipid asymmetry across membranes of late secretory and endocytic compartments, but their kinship to cation-transporting P-type transporters raised doubts about whether P(4)-ATPases alone are sufficient to mediate flippase activity. P(4)-ATPases form heteromeric complexes with Cdc50 proteins. Studies of the enzymatic properties of purified P(4)-ATPase·Cdc50 complexes showed that catalytic activity depends on direct and specific interactions between Cdc50 subunit and transporter, whereas in vivo interaction assays suggested that the binding affinity for each other fluctuates during the transport reaction cycle. The structural determinants that govern this dynamic association remain to be established. Using domain swapping, site-directed, and random mutagenesis approaches, we here show that residues throughout the subunit contribute to forming the heterodimer. Moreover, we find that a precise conformation of the large ectodomain of Cdc50 proteins is crucial for the specificity and functionality to transporter/subunit interactions. We also identified two highly conserved disulfide bridges in the Cdc50 ectodomain. Functional analysis of cysteine mutants that disrupt these disulfide bridges revealed an inverse relationship between subunit binding and P(4)-ATPase-catalyzed phospholipid transport. Collectively, our data indicate that a dynamic association between subunit and transporter is crucial for the transport reaction cycle of the heterodimer.     

Regulation of connexin‐ and pannexin‐based channels by post‐translational modifications

Connexin (Cx) and pannexin (Panx) proteins form large conductance channels, which function as regulators of communication between neighbouring cells via gap junctions and/or hemichannels. Intercellular communication is essential to coordinate cellular responses in tissues and organs, thereby fulfilling an essential role in the spreading of signalling, survival and death processes. The functional properties of gap junctions and hemichannels are modulated by different physiological and pathophysiological stimuli. At the molecular level, Cxs and Panxs function as multi‐protein channel complexes, regulating their channel localisation and activity. In addition to this, gap junctional channels and hemichannels are modulated by different post‐translational modifications (PTMs), including phosphorylation, glycosylation, proteolysis, N‐acetylation, S‐nitrosylation, ubiquitination, lipidation, hydroxylation, methylation and deamidation. These PTMs influence almost all aspects of communicating junctional channels in normal cell biology and pathophysiology. In this review, we will provide a systematic overview of PTMs of communicating junction proteins and discuss their effects on Cx and Panx‐channel activity and localisation.     

Connexins: sensors and regulators of cell cycling

It is nowadays well established that gap junctions are critical gatekeepers of cell proliferation, by controlling the intercellular exchange of essential growth regulators. In recent years, however, it has become clear that the picture is not as simple as originally anticipated, as structural precursors of gap junctions can affect cell cycling by performing actions not related to gap junctional intercellular communication. Indeed, connexin hemichannels also foresee a pathway for cell growth communication, albeit between the intracellular compartment and the extracellular environment, while connexin proteins as such can directly or indirectly influence the production of cell cycle regulators independently of their channel activities. Furthermore, a novel set of connexin-like proteins, the pannexins, have lately joined in as regulators of the cell proliferation process, which they can affect as either single units or as channel entities. In the current paper, these multifaceted aspects of connexin-related signalling in cell cycling are reviewed.     

Cdc50p Plays a Vital Role in the ATPase Reaction Cycle of the Putative Aminophospholipid Transporter Drs2p

Members of the P₄ subfamily of P-type ATPases are believed to catalyze transport of phospholipids across cellular bilayers. However, most P-type ATPases pump small cations or metal ions, and atomic structures revealed a transport mechanism that is conserved throughout the family. Hence, a challenging problem is to understand how this mechanism is adapted in P₄-ATPases to flip phospholipids. P₄-ATPases form heteromeric complexes with Cdc50 proteins. The primary role of these additional polypeptides is unknown. Here, we show that the affinity of yeast P₄-ATPase Drs2p for its Cdc50-binding partner fluctuates during the transport cycle, with the strongest interaction occurring at a point where the enzyme is loaded with phospholipid ligand. We also find that specific interactions with Cdc50p are required to render the ATPase competent for phosphorylation at the catalytically important aspartate residue. Our data indicate that Cdc50 proteins are integral components of the P₄-ATPase transport machinery. Thus, acquisition of these subunits may have been a crucial step in the evolution of flippases from a family of cation pumps.P-type ATPases form a large family of membrane pumps that are transiently autophosphorylated at a conserved aspartate residue, hence the designation P-type. Prominent examples include the Ca²⁺-ATPase SERCA,⁴ which pumps Ca²⁺ from the cytosol into the lumen of the sarcoplasmic reticulum of skeletal muscle cells (1), and the Na⁺/K⁺-ATPase, which generates the electrochemical gradients for sodium and potassium that are vital to animal cells (2). Transport is accomplished by cyclic changes between two main enzyme conformations, E₁ and E₂, during which the ATPase is phosphorylated by ATP at the aspartate residue and subsequently dephosphorylated. These processes are coupled to vectorial transport and counter-transport by a controlled opening and closing of cytoplasmic and exoplasmic pathways, which give access to the ion-binding sites that are buried inside the membrane-spanning region of the pump (3). A host of crystal structures of the Ca²⁺ pump SERCA in well defined states of the reaction cycle revealed important aspects of the transport mechanism (4, 5). Sequence homology and structures of other ATPases show that this mechanism rests on principles and structural elements that apply to all P-type ATPases (6–8).Although P-type ATPases usually pump small cations or metal ions, members of the P₄ subfamily form a notable exception. A growing body of evidence indicates that P₄-ATPases catalyze phospholipid transport and create membrane lipid asymmetry (9–11). This process contributes to a multitude of cellular functions, including membrane vesiculation, cell division, and life span. The yeast Saccharomyces cerevisiae contains five P₄-ATPases, namely Dnf1p and Dnf2p at the plasma membrane, Drs2p and Dnf3p in the trans-Golgi network, and Neo1p in an endosomal compartment (12–14). Removal of Dnf1p and Dnf2p abolishes inward translocation of 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))-labeled analogs of phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylcholine (PC) and causes an aberrant exposure of endogenous aminophospholipids at the cell surface (13, 15). Trans-Golgi membranes isolated from a yeast strain that lacks the Dnf proteins and contains a temperature-sensitive drs2 allele display a defect in 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))-PS translocation when shifted to the non-permissive temperature (16). The latter finding provides strong evidence that Drs2p is directly coupled to flippase activity, and subsequent studies showed that Drs2p, together with Dnf3p, are required for maintaining PE asymmetry in post-Golgi secretory vesicles (17).Although no P₄-ATPase has been shown to display flippase activity in reconstitution experiments with purified enzyme, the relationship of P₄-ATPases to flippase activity and lipid asymmetry has gained further support from functional studies in various other organisms, including parasites (18), plants (19), worms (20), and mice (21). Besides a common domain organization, P₄-ATPases display a clear sequence homology with cation-transporting P-type pumps. Shared sequence motifs include the canonical phosphorylation site in the P domain, the nucleotide-binding site in the N domain, and a TGES-related sequence in the A domain (22). This implies that P₄-ATPases and cation pumps use the same mechanism to couple ATP hydrolysis to ligand transport. Phospholipid transport by P₄-ATPases would correspond to counter-transport of H⁺ ions by the Ca²⁺ pump and of K⁺ ions by the Na⁺/K⁺-ATPase as the direction of movement is from the exoplasmic to the cytoplasmic leaflet. During the reaction cycle of cation pumps, access to the ion-binding pocket alternates between the two sides of the membrane, with the ions becoming temporarily occluded after each ion binding event (23). How this mechanism is adapted in P₄-ATPases to translocate phospholipids is unclear. Flippases must provide a sizeable hydrophilic pathway for the polar headgroup to pass through the membrane as well as accommodate the hydrophobic nature of the lipid backbone. Whether P₄-ATPases alone are sufficient to accomplish this task is not known.Recent studies revealed that P₄-ATPases form complexes with members of the Cdc50 protein family (24). Cdc50 proteins consist of two membrane spans and a large, N-glycosylated ectodomain with one or more conserved disulfide bonds (25). The yeast family members Cdc50p, Lem3p, and Crf1p can be co-immunoprecipitated with Drs2p, Dnf1p/Dnf2p, and Dnf3p, respectively. Formation of these complexes is required for proper expression and endoplasmic reticulum (ER) export of either partner (24, 26) so that mutation of one member of the complex phenocopies mutations in the other (15, 25). This behavior in yeast is mirrored in other organisms; Ld Ros3, a Lem3p homolog in Leishmania parasites, is needed for proper trafficking of the P₄-ATPase Ld MT (18), whereas the human P₄-ATPase ATP8B1 requires a Cdc50p homolog, CDC50A, for ER exit and delivery to the plasma membrane (27). Moreover, the Arabidopsis P₄-ATPase ALA3 requires its Cdc50-binding partner ALIS1 to complement the lipid transport defect at the plasma membrane in a Δdnf1Δdnf2Δdrs2 yeast mutant (19).Together, the above findings indicate that Cdc50 subunits are indispensable for a proper functioning of P₄-ATPases and that it is the combination of the two that yields a physiologically active transporter. However, these studies have not clarified the primary function of the Cdc50 polypeptide in the complex. Here, we provide the first evidence that Cdc50 subunits play a crucial role in the P₄-ATPase reaction cycle. Using a genetic reporter system, we find that P₄-ATPase-Cdc50 interactions are dynamic and tightly coupled to the ATPase reaction cycle. Moreover, by characterizing the enzymatic properties of a purified P₄-ATPase-Cdc50 complex, we show that catalytic activity relies on direct and specific interactions between the subunit and transporter.     

Pannexin channels in ATP release and beyond: An unexpected rendezvous at the endoplasmic reticulum

The pannexin (Panx) family of proteins, which is co-expressed with connexins (Cxs) in vertebrates, was found to be a new GJ-forming protein family related to invertebrate innexins. During the past ten years, different studies showed that Panxs mainly form hemichannels in the plasma membrane and mediate paracrine signalling by providing a flux pathway for ions such as Ca²⁺, for ATP and perhaps for other compounds, in response to physiological and pathological stimuli. Although the physiological role of Panxs as a hemichannel was questioned, there is increasing evidence that Panx play a role in vasodilatation, initiation of inflammatory responses, ischemic death of neurons, epilepsy and in tumor suppression. Moreover, it is intriguing that Panxs may also function at the endoplasmic reticulum (ER) as intracellular Ca²⁺-leak channel and may be involved in ER-related functions. Although the physiological significance and meaning of such Panx-regulated intracellular Ca²⁺ leak requires further exploration, this functional property places Panx at the centre of many physiological and pathophysiological processes, given the fundamental role of intracellular Ca²⁺ homeostasis and dynamics in a plethora of physiological processes. In this review, we therefore want to focus on Panx as channels at the plasma membrane and at the ER membranes with a particular emphasis on the potential implications of the latter in intracellular Ca²⁺ signalling.     

Pannexins,distant relatives of the connexin family with specific cellular functions?

Intercellular communication (IC) is mediated by gap junctions (GJs) and hemichannels, which consist of proteins. This has been particularly well documented for the connexin (Cx) family. Initially, Cxs were thought to be the only proteins capable of GJ formation in vertebrates. About 10 years ago, however, a new GJ‐forming protein family related to invertebrate innexins (Inxs) was discovered in vertebrates, and named the pannexin (Panx) family. Panxs, which are structurally similar to Cxs, but evolutionarily distinct, have been shown to be co‐expressed with Cxs in vertebrates. Both protein families show distinct properties and have their own particular function. Identification of the mechanisms that control Panx channel gating is a major challenge for future work. In this review, we focus on the specific properties and role of Panxs in normal and pathological conditions.     

Mechanism and significance of P4 ATPase-catalyzed lipid transport: Lessons from a Na/K-pump

Members of the P₄ subfamily of P-type ATPases are believed to catalyze phospholipid transport across membrane bilayers, a process influencing a host of cellular functions. Atomic structures and functional analysis of P-type ATPases that pump small cations and metal ions revealed a transport mechanism that appears to be conserved throughout the family. A challenging problem is to understand how this mechanism is adapted in P₄ ATPases to flip phospholipids. P₄ ATPases form oligomeric complexes with members of the CDC50 protein family. While formation of these complexes is required for P₄ ATPase export from the endoplasmic reticulum, little is known about the functional role of the CDC50 subunits. The Na⁺/K⁺-ATPase and closely-related H⁺/K⁺-ATPase are the only other P-type pumps that are oligomeric, comprising mandatory β-subunits that are strikingly reminiscent of CDC50 proteins. Besides serving a role in the functional maturation of the catalytic α-subunit, the β-subunit also contributes specifically to intrinsic transport properties of the Na⁺/K⁺ pump. As β-subunits and CDC50 proteins likely adopted similar structures to accomplish analogous tasks, current knowledge of the Na⁺/K⁺-ATPase provides a useful guide for understanding the inner workings of the P₄ ATPase class of lipid pumps.