Corresponding Author: Francisco J. López-Hernández
Institute of Biomedical Research of Salamanca (IBSAL), University of Salamanca, Edificio Departamental, S-20, Campus Miguel de Unamuno, 37007 Salamanca (Spain)
Tel. +34 923 294500 # 1444 , E-Mail firstname.lastname@example.org
Cell Surface Area to Volume Relationship During Apoptosis and Apoptotic Body Formation
Francisco J. López-Hernándeza,b,c,d,e
aInstitute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain, bFundación Instituto de Estudios de Ciencias de la Salud de Castilla y León (IECSCYL), Soria, Spain, cDepartment of Physiology and Pharmacology. University of Salamanca (USAL), Salamanca, Spain, dGroup of Translational Research on Renal and Cardiovascular Diseases (TRECARD), Salamanca, Spain. National Network for Kidney Research REDINREN, RD016/0009/0025, Instituto de Salud Carlos III, Madrid, Spain, eGroup of Biomedical Research on Critical Care (BioCritic), Valladolid, Spain
Cell volume regulation is a tightly controlled, continuous and dynamic process that is essential for cellular homeostasis, integrity and normal function . Unless the cell undergoes growth or division, cell volume must be maintained constant to allow physiological performance and cell integrity . In such circumstances, swelling or shrinkage leads to dysfunction and sterile death. However, in specific scenarios, such as during apoptotic programmed cell death, cell volume varies both as a necessary and a permissive event . Specifically, inhibition of volume decrease early halts the apoptotic process . Governed by regulated membrane channels and transporters, cell volume is dictated by the transmembrane ionic flux equilibrium that drags water in or out of the cell following the osmotic gradient, whereas plasmalemmal stretch and tension have minimal impact on cytosolic pressure and thus on volume regulation . Many studies have addressed the mechanisms implicated in cell volume regulation under different biological scenarios. In this brief article, we analyze, from a geometric point of view, the changes in cell volume in connection and coordination with cell and plasma membrane surface areas during apoptosis. Accommodation of cell volume to available membrane area is an inextricable determinant of this process, which will be analyzed in the following sections.
Apoptosis: from apoptotic volume decrease to disintegration into apoptotic bodies
Apoptosis is a programmed form of cell death. Apoptotic (or apoptosis-resembling) programs are found in most living beings, from some unicellular organisms to vertebrates [6–9]. It is believed that programmed cell death has played evolving roles through evolution (in embryogenesis, homeostasis, disease prevention and altruism) . Thus, the apoptotic phenotype has also evolved accordingly to accommodate increasing biological demands . For instance, an ultimate event in the apoptotic program is the dismantling of the dying cell into smaller, membrane bound vesicles, known as apoptotic bodies (ABs). ABs contain an aliquot of all cell constituents (and the debris generated from their controlled degradation) packed and sealed by intact plasma membrane fragments .
Cell dismantling into ABs is believed to pursue two specific biological goals in pluricellular organisms endowed with an immune system. On the one hand, easier engulfment and disposal by neighboring and immune system cells. On the other hand, prevention of immunological overstimulation that would result from an uncontrolled released of cell content. This is opposed to other forms of cell death characterized by swelling and plasmalemmal rupture . Shedding of cell content to the interstitium or extracellular space is a strong immunological activator. Many cell components (i.e. specific molecules from all structures and
organelles)  are known damage-associated molecular patterns (DAMPs). DMAPs intensely attract immune system cells and strongly activate the innate response leading to inflammation . The immune and inflammatory response is a double-edged phenomenon. Even under controlled circumstances, this response has significant side-effects and energetic cost. When exacerbated or abnormally extended in time or in its natural evolution and resolution, the immune response results in severe tissue-injuring effects.
Cell dismantling into ABs is an essential and distinctive characteristic that biologically differentiates apoptosis from other cell death modes. All other phenomena observed during the initiation and execution of apoptosis serve the correct achievement of this final purpose. These phenomena prominently include the very early event known as apoptotic volume decrease (AVD), a process that reduces cell volume . AVD, a universal characteristic of apoptotic cells , is not a passive consequence secondary to other events, but an active process with switch properties on the apoptotic program. Inhibition of AVD early halts the execution of apoptosis and thus inhibits cell death and AB formation [17–20]. AVD results from water extrusion osmotically driven out of the cell by net K+ and Cl- efflux . In agreement with this, caspases (i.e. the core enzymes of apoptosis) are inactive at the normal intracellular concentration of K+. Caspases are activated by cleavage of their zymogens, but they also require a low K+ environment to function [18, 22]. Other components of the apoptotic machinery, such as nucleases responsible for DNA fragmentation [18, 22], and formation of the apoptosome  are also inhibited by normal K+ concentration, and are only allowed to occur upon AVD.
Two consecutive waves of AVD have been shown to occur in some cell types, with distinct roles in the process of apoptosis [24–26]. In the early stage, Na+ and K+ gradients are reversed, resulting in intracellular accumulation of Na+ and K+ extrusion, and in a 20–40% drop of the cell volume taking place before mitochondrial cytochrome c release . In a second round, cell volume further shrinks leveraging additional K+ outflow. This second stage is dependent on cytochrome c release and caspase activation and, interestingly, also on proper cytoskeleton organization . In fact, cytoskeleton-disrupting agents prevent this second round of AVD, along with DNA degradation and formation of ABs [24, 27]. A channel-mediated K+ outward movement activated by cytochrome c independently of initiator caspase-9 activation is involved [28[. However, channels behind AVD might be different from one cell type to another. Some channel redundancy is thought to ensure AVD under a variety of conditions, reinforcing the importance of AVD in apoptosis [18–20].
The process of cell dismantling into ABs not only occurs in the (classical) way depicted in Fig. 1. Newly identified forms involve highly regulated processes of cell projections from which one or multiple smaller ABs detach [29–31]. In fact, as shown in Fig. 1, cells sometimes divide into a smaller number of larger ABs of diverse sizes [32–37], and also into smaller ABs resulting from other, recently described processes of disassembly. These processes occur through structures known as apoptopodia, beaded apoptopodia [29, 31] and microtubule spikes, from a larger corpse . The mechanism of cell disassembly determines AB size [29–31].
AB formation is a complex process regulated by coordinated morphological steps including apoptotic membrane blebbing (or zeiosis), protrusion formation, and eventual fragmentation [14, 30]. Blebs are formed when the plasma membrane delaminates from the cortical cytoskeletal network at specific locations. This process (as most apoptotic events) is orchestrated by caspases . In fact, delamination occurs because of retraction of the actin-myosin II cortex (in a caspase and Rho-dependent manner). Delamination significantly alters membrane dynamics, thus facilitating the formation of membrane blebs [38, 39]. At delamination sites, membrane blisters are formed and expanded by the increased hydrostatic pressure produced by actomyosin-mediated cellular contraction. The size of blebs gradually increases during the progression of apoptosis, eventually forming large vesicles  that subsequently separate from the main cell body to form ABs [14, 41, 42]. At the final stages of apoptosis the actin cytoskeleton is degraded and phagocytosis of the apoptotic bodies ensues .
The author declares no conflict of interests exist.
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