The Cytoskeleton and Its Regulation by Calcium and Protons

Calcium and protons exert control over the formation and activity of the cytoskeleton, usually by modulating an associated motor protein or one that affects the structural organization of the polymer.The cytoskeleton is well recognized as an ever-present component of all eukaryotic cells; more recently, it has been identified in prokaryotic cells as well (van den Ent et al., 2001; Wickstead and Gull, 2011). The cytoskeleton gives order to a cell, and in large cells, where diffusion may become limiting, it provides a means to move components around, thus facilitating reactions (Verchot-Lubicz and Goldstein, 2010). Increasingly, we also see that the cytoskeleton in eukaryotic cells participates in the uptake of material from outside of the cell (e.g. endocytosis and phagocytosis; Yutin et al., 2009). And inside the cell, where organelles, membrane systems, and macromolecular complexes are exquisitely and dynamically organized in minute detail, it is largely the cytoskeleton that is responsible for this organization. Because the cytoskeleton participates in so many processes, it becomes a matter of consequence to understand how it is controlled. Indeed, this is a topic of major interest at present and one whose solution will contribute fundamentally to our understanding of many aspects of cell growth and development.Among several possible control elements, it has been widely known for many years that ions, in particular calcium, can exert a profound effect on the structure and activity of both the actomyosin and tubulin cytoskeletons (Hepler, 2005; Hepler and Winship, 2015). One of the best examples is the stimulation of the contraction of striated muscle by calcium, where, through its binding to troponin C, tropomyosin is displaced along the actin filament, exposing myosin-binding sites and permitting contraction to occur (Alberts et al., 2008). There are numerous other examples found in both plant and animal cells involving calcium regulation of the actin cytoskeleton. In addition, it is also well established that calcium can have profound effects on microtubules (MTs). Indeed, the demonstration in 1972 by Richard Weisenberg that elevated calcium caused MT depolymerization was transformative (Weisenberg, 1972). Whereas biochemists until that time had been unsuccessful in obtaining in vitro polymerization of MTs, this now became possible. But for me, it raised intriguing possibilities concerning a role for calcium in the control of cellular processes such as mitosis and cytokinesis (Hepler and Wayne, 1985; Hepler, 2005). It also got me to think more widely about the role of calcium as a general signaling agent. In the 30 years since Randy Wayne and I reviewed this topic (Hepler and Wayne, 1985), it is now apparent that calcium reaches into countless events and processes and can be viewed as a universal signaling agent in plant cell growth and development (Edel and Kudla, 2015).In advance, I must tell you that I will not review the broad scope of calcium research today, which is vast; there are many reviews to which the interested reader is directed (Hetherington and Brownlee, 2004; Kudla et al., 2010; Verret et al., 2010; Hashimoto and Kudla, 2011; Hamel et al., 2014; Edel and Kudla, 2015). I will also not discuss the role of small GTPase proteins, even though they can have profound effects on calcium and the cytoskeleton in plant cells. Again, the interested reader is directed to pertinent reviews on this topic (Gu et al., 2005; Nibau et al., 2006; Craddock et al., 2012; Oda and Fukuda, 2013; Li et al., 2015). Rather, in this article, I will focus on the role of ions in the control of the cytoskeleton in plant cells, giving attention to those actin- and tubulin-binding proteins that are modulated by calcium and/or protons. Before discussing ion regulation, I briefly consider, first, how calcium and protons emerged as signaling agents and, second, aspects of cytoskeleton evolution in the progression from prokaryotic to eukaryotic cells.     

Bud formation inFunaria: Organelle redistribution following cytokinin treatment

Summary Cytokinin stimulates caulonemata ofFunaria to undergo an asymmetric division leading to the gametophore. The earliest detectable event is a small protuberance at the distal portion of the cell accompanied by the reorganization of the underlying organelles into a polarized distribution reminiscent of a tip growing cell. Dictyosomes and associated vesicles accumulate in the protuberance directly beneath the plasma membrane with mitochondria subjacent to the vesicular layer. Endoplasmic reticulum lies beneath the mitochondrial zone directly above the large central vacuole, while chloroplasts are outside the bud. As development continues the bud elongates causing the outer cell wall to exfoliate. During the above events the nucleus migrates toward the bud site concomitant with an increase in the number of microtubules between the nucleus and the base of the outgrowth. Nucleoli, extruded from the nucleus during a previous division, persist as diffuse fragments within the protuberance. Upon reaching the bud site, division occurs with the developing phragmoplast being initiated distal to the caulonema tip cell. The former polarized distribution of the cytoplasm is altered as mitochondria, chloroplasts and small vacuoles become evenly dispersed throughout the cytoplasm; dicytosomes and endoplasmic reticulum occupy a cortical position. These events indicate a change from 2-D tip growth to 3-D diffuse growth. To quantify the ultrastructural changes associated with bud formation we performed a morphometric analysis of cells in various stages of budding. The relative volumes of dictyosomes and vesicles adjacent to the bud apex decrease during bud development coincident with an increase in these organelles in lower portions of the cytoplasm. Mitochondria and chloroplasts follow this same pattern although their highest relative volumes initially are 4 m from the bud apex and outside the bud site, respectively. These data, as well as density profile topographic maps for vesicle fractions, support the contention that cytokinin induces a change in morphological symmetry and polarity in the fine structure ofFunaria.     

Epidermal growth factor (EGF) and hormones stimulate phosphoinositide hydrolysis and increase EGF receptor protein synthesis and mRNA levels in rat liver epithelial cells. Evidence for protein kinase C-dependent and -independent pathways

Epidermal growth factor (EGF) stimulated the rapid accumulation of inositol trisphosphate in WB cells, a continuous line of rat hepatic epithelial cells. Since we previously had shown that EGF stimulates EGF receptor synthesis in these cells, we tested whether hormones that stimulate PtdIns(4,5)P2 hydrolysis would increase EGF receptor protein synthesis and mRNA levels. Epinephrine, angiotensin II, and [Arg8]vasopressin activate phospholipase C in WB cells as evidenced by the accumulation of the inositol phosphates, inositol monophosphate, inositol bisphosphate, and inositol trisphosphate. A 3-4-h treatment with each hormone also increased the rate of EGF receptor protein synthesis by 3-6-fold as assessed by immunoprecipitation of EGF receptor from [35S]methionine-labeled cells. Northern blot analyses of WB cell EGF receptor mRNA levels revealed that agents linked to the phosphoinositide signaling system increased receptor mRNA content within 1-2 h. A maximal increase of 3-7-fold was observed after a 3-h exposure to EGF and hormones. The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), which activates protein kinase C also stimulated EGF receptor synthesis. Pretreatment of WB cells for 18 h with high concentrations of TPA "down-regulated" protein kinase C and blocked TPA-directed EGF receptor mRNA synthesis. In contrast, the effect of EGF on EGF receptor mRNA levels was not significantly decreased by TPA pretreatment. Epinephrine-induced increases in EGF receptor mRNA were reduced from 4- to 2-fold. Similarly, 18 h TPA pretreatment abolished the effect of TPA on EGF receptor protein synthesis but did not affect EGF-dependent EGF receptor protein synthesis. The 18-h TPA pretreatment diminished by 30-50% the induction of receptor protein synthesis by epinephrine or angiotensin II. We conclude that in WB cells EGF receptor synthesis can be regulated by EGF and other hormones that stimulate PtdIns(4,5)P2 hydrolysis. In these cells, EGF receptor synthesis appears to be regulated by several mechanism: one pathway is dependent upon EGF receptor activation and can operate independently of protein kinase C activation; another pathway is correlated with PtdIns(4,5)P2 hydrolysis and is dependent, at least in part, upon protein kinase C activation.     

Rhizobium Nod factors induce increases in intracellular free calcium and extracellular calcium influxes in bean root hairs

Application of Nod factors to growing, responsive root hairs of the bean Phaseolus vulgaris induces marked changes in both the intracellular cytosolic free calcium (Ca²⁺) and in the influx of extracellular [Ca²⁺]. The intracellular [Ca²⁺], which has been measured by ratiometric imaging in cells microinjected with fura-2-dextran (70 kDa), elevates within 5 min from approximately 400 n m to 1500 n m in localised zones in the root hair apex. Of particular note is the observation that the elevated regions of [Ca²⁺] appear to shift position during short time intervals. Increases in and fluctuations of the intracellular [Ca²⁺] are also observed in the perinuclear region after 10–15 min treatment with Nod factors. The extracellular Ca²⁺ flux, detected with the non-invasive, calcium specific vibrating electrode, is inwardly directed and also increases quickly in response to Nod factors from 13 pmol cm^–2 s^–1 to 28 pmol cm^–2 s^–1. Chitin-oligomers, which are structurally similar but biologically inactive when compared to the active Nod factors, fail to elicit changes in either intracellular or extracellular Ca²⁺. The similar timing and location of the intracellular elevations and the increased extracellular influx provide support for the idea that Ca²⁺ participates in secretion and cell wall remodelling, which occur in anticipation of root hair deformation and curling.     

Uncoupling secretion and tip growth in lily pollen tubes: evidence for the role of calcium in exocytosis

Cytoplasmic calcium concentration ([Ca²⁺]_i) and extracellular calcium (Ca²⁺_o) influx has been studied in pollen tubes of Lilium longliflorum in which the processes of cell elongation and exocytosis have been uncoupled by use of Yariv phenylglycoside ((β-D-Glc)₃). Growing pollen tubes were pressure injected with the ratio dye fura-2 dextran and imaged after application of (β-D-Glc)₃, which binds arabinogalactan proteins (AGPs). Application of (β-D-Glc)₃ inhibited growth but not secretion. Ratiometric imaging of [Ca²⁺]_i revealed an initial spread in the locus of the apical [Ca²⁺]_i gradient and substantial elevations in basal [Ca²⁺]_i followed by the establishment of new regions of elevated [Ca²⁺]_i on the flanks of the tip region. Areas of elevated [Ca²⁺]_i corresponded to sites of pronounced exocytosis, as evidenced by the formation of wall ingrowths adjacent to the plasma membrane. Ca²⁺_o influx at the tip of (β-D-Glc)₃-treated pollen tubes was not significantly different to that of control tubes. Taken together these data indicate that regions of elevated [Ca²⁺]_i, probably resulting from Ca²⁺_o influx across the plasma membrane, stimulate exocytosis in pollen tubes independent of cell elongation.