Rop GTPase-dependent dynamics of tip-localized F-actin controls tip growth in pollen tubes

Tip-growing pollen tubes provide a useful model system to study polar growth. Although roles for tip-focused calcium gradient and tip-localized Rho-family GTPase in pollen tube growth is established, the existence and function of tip-localized F-actin have been controversial. Using the green fluorescent protein-tagged actin-binding domain of mouse talin, we found a dynamic form of tip-localized F-actin in tobacco pollen tubes, termed short actin bundles (SABs). The dynamics of SABs during polar growth in pollen tubes is regulated by Rop1At, a Rop GTPase belonging to the Rho family. When overexpressed, Rop1At transformed SAB into a network of fine filaments and induced a transverse actin band behind the tip, leading to depolarized growth. These changes were due to ectopic Rop1At localization to the apical region of the plasma membrane and were suppressed by guanine dissociation inhibitor overexpression, which removed ectopically localized Rop1At. Rop GTPase-activating protein (RopGAP1) overexpression, or Latrunculin B treatments, also recovered normal actin organization and tip growth in Rop1At-overexpressing tubes. Moreover, overexpression of RopGAP1 alone disrupted SABs and inhibited growth. Finally, SAB oscillates and appears at the tip before growth. Together, these results indicate that the dynamics of tip actin are essential for tip growth and provide the first direct evidence to link Rho GTPase to actin organization in controlling cell polarity and polar growth in plants.     

Regulation of the tip-high [Ca2+] gradient in growing hyphae of the fungus Neurospora crassa

Previous work has shown that hyphal elongation in the fungus Neurospora crassa requires a tip-high cytosolic Ca2+ gradient. The source of the Ca2+ appears to be intracellular stores as there is no net transplasma membrane Ca2+ flux at the elongating hyphal tip and modification of ion fluxes across the plasma membrane using voltage clamp is without effect on tip growth. To decode the internal mechanisms which generate and maintain the tip-high Ca2+ gradient we first identified calcium regulators which affect hyphal growth and morphology, then determined how they modify cytosolic [Ca2+] and the actin cytoskeleton using fluorescent dyes and confocal microscopy. Cyclopiazonic acid (a known inhibitor of the endoplasmic reticulum calcium ATPase) inhibits growth and increases cytoplasmic [Ca2+] in the basal region 10-25 microm behind the hyphal tip. 2-APB (2-aminoethoxydiphenyl borate, an inhibitor of IP3-induced Ca2+ release) inhibits hyphal elongation and dissipates the tip-high Ca2 gradient 0-10 microm from the tip. Microinjections of the IP3 receptor agonists adenophostin A and IP3 (but not control microinjections of the biologically inactive L-IP3) transiently inhibited growth and induced subapical branches. IP3 microinjections, but not L-IP3, lowered tip-localized [Ca2+] and increased basal [Ca2+]. Even though their effect on [Ca2+] gradients was different, both cyclopiazonic acid and 2-APB disrupted similarly the normal actin pattern at the hyphal apex. Conversely, disruption of actin with latrunculin B dissipated tip-localized Ca2+. We conclude that the tip-high Ca2+ gradient is generated internally by Ca2+ sequestration into endoplasmic reticulum behind the tip and Ca2+ release via an IP3 receptor from tip-localized vesicles whose location is maintained by the actin cytoskeleton.     

Targeting and Regulation of Cell Wall Synthesis During Tip Growth in Plants

Root hairs and pollen tubes are formed through tip growth, a process requiring synthesis of new cell wall material and the precise targeting and integration of these components to a selected apical plasma membrane domain in the growing tips of these cells. Presence of a tip-focused calcium gradient, control of actin cytoskeleton dynamics, and formation and targeting of secretory vesicles are essential to tip growth. Similar to cells undergoing diffuse growth, cellulose, hemicelluloses, and pectins are also deposited in the growing apices of tip-growing cells. However, differences in the manner in which these cell wall components are targeted and inserted in the expanding portion of tip-growing cells is reflected by the identification of elements of the plant cell wall synthesis machinery which have been shown to play unique roles in tip-growing cells. In this review, we summarize our current understanding of the tip growth process, with a particular focus on the subcellular targeting of newly synthesized cell wall components, and their roles in this form of plant cell expansion.     

The ROP2 GTPase controls the formation of cortical fine F-actin and the early phase of directional cell expansion during Arabidopsis organogenesis

Polar cell expansion in differentiating tissues is critical for the development and morphogenesis of plant organs and is modulated by hormonal and developmental signals, yet little is known about signaling in this fundamental process in plants. In contrast to tip-growing cells, such as pollen tubes and root hairs, cells in developing tissues are thought to expand by diffuse growth. In this study, we provide evidence that these cells expand in two phases with distinct mechanisms. In the early phase, cell expansion can occur in both longitudinal and radial or lateral directions and is mediated by Rop GTPase signaling, a mechanism known to control tip growth. The expression of a dominant-negative mutant for ROP2 (DN-rop2) inhibited polar cell expansion, whereas the expression of a constitutively active mutant (CA-rop2) caused isotropic expansion in the early phase. In the late phase, expansion occurs only in the longitudinal direction and is not affected by DN-rop2 or CA-rop2 expression. The transition from the early to the late phase coincides with the reorientation of cortical microtubules from random to transverse arrangements. Thus, cell expansion in the late phase is consistent with polar diffuse growth, in which polarity probably is defined by transverse cortical microtubules. We show that the direction of cell expansion in the early phase is associated with the localization of diffuse fine cortical F-actin in leaf epidermal cells. DN-rop2 expression specifically inhibited the formation of this F-actin, but not actin cables, whereas CA-rop2 expression caused delocalized distribution of this fine F-actin throughout the cell cortex. Furthermore, green fluorescent protein-ROP2 was localized preferentially to the cortical region of the cell, where expansion apparently occurs. These observations suggest that ROP2 control of the polar expansion of cells within tissues is analogous to the Rop control of tip growth and of tip-localized F-actin in pollen tubes and root hairs and that the tip growth mechanism also may modulate polar cell expansion in differentiating tissues.     

A Rho family GTPase controls actin dynamics and tip growth via two counteracting downstream pathways in pollen tubes

Tip growth in neuronal cells, plant cells, and fungal hyphae is known to require tip-localized Rho GTPase, calcium, and filamentous actin (F-actin), but how they interact with each other is unclear. The pollen tube is an exciting model to study spatiotemporal regulation of tip growth and F-actin dynamics. An Arabidopsis thaliana Rho family GTPase, ROP1, controls pollen tube growth by regulating apical F-actin dynamics. This paper shows that ROP1 activates two counteracting pathways involving the direct targets of tip-localized ROP1: RIC3 and RIC4. RIC4 promotes F-actin assembly, whereas RIC3 activates Ca(2+) signaling that leads to F-actin disassembly. Overproduction or depletion of either RIC4 or RIC3 causes tip growth defects that are rescued by overproduction or depletion of RIC3 or RIC4, respectively. Thus, ROP1 controls actin dynamics and tip growth through a check and balance between the two pathways. The dual and antagonistic roles of this GTPase may provide a unifying mechanism by which Rho modulates various processes dependent on actin dynamics in eukaryotic cells.     

Differential mitochondrial calcium responses in different cell types detected with a mitochondrial calcium fluorescent indicator, mito-GCaMP2

Mitochondrial calcium plays a crucial role in mitochondrial metabolism, cell calcium handling, and cell death. However, some mechanisms concerning mitochondrial calcium regulation are still unknown, especially how mitochondrial calcium couples with cytosolic calcium. In this work, we constructed a novel mitochondrial calcium fluorescent indicator (mito-GCaMP2) by genetic manipulation. Mito-GCaMP2 was imported into mitochondria with high efficiency and the fluorescent signals co-localized with that of tetramethyl rhodamine methyl ester, a mitochondrial membrane potential indicator. The mitochondrial inhibitors specifically decreased the signals of mito-GCaMP2. The apparent K(d) of mito-GCaMP2 was 195.0 nmol/L at pH 8.0 in adult rat cardiomyocytes. Furthermore, we observed that mito-GCaMP2 preferred the alkaline pH surrounding of mitochondria. In HeLa cells, we found that mitochondrial calcium ([Ca(2+)](mito)) responded to the changes of cytosolic calcium ([Ca(2+)](cyto)) induced by histamine or thapasigargin. Moreover, external Ca(2+) (100 μmol/L) directly induced an increase of [Ca(2+)](mito) in permeabilized HeLa cells. However, in rat cardiomyocytes [Ca(2+)](mito) did not respond to cytosolic calcium transients stimulated by electric pacing or caffeine. In permeabilized cardiomyocytes, 600 nmol/L free Ca(2+) repeatedly increased the fluorescent signals of mito-GCaMP2, which excluded the possibility that mito-GCaMP2 lost its function in cardiomyocytes mitochondria. These results showed that the response of mitochondrial calcium is diverse in different cell lineages and suggested that mitochondria in cardiomyocytes may have a special defense mechanism to control calcium flux.     

Regulation of the mitochondrial adenine nucleotide pool size in liver: mechanism and metabolic role

The ATP-Mg/Pi carrier in liver mitochondria can catalyze the exchange of ATP-Mg on one side of the inner membrane for Pi on the other. This mechanism allows for net uptake or release of ATP-Mg from mitochondria and thus regulates the matrix ATP + ADP + AMP pool size. In isolated mitochondria, carrier activity is stimulated by submicromolar concentrations of calcium, suggesting that calcium may regulate transport rates in vivo. Whenever the carrier is active, the direction of any net changes in the matrix adenine nucleotide pool size is determined mainly by the extent to which the prevailing ATP-Mg concentration gradient deviates from an equilibrium related to delta pH through the phosphate concentration gradient. Thus it seems that in the cell, energy status (reflected by ATP:ADP ratios in the cytoplasm and matrix) determines whether calcium-mediated hormone activation of the carrier will produce an increase or a decrease in the matrix adenine nucleotide content. Consequent variations in the absolute concentrations of ATP, ADP, and AMP in the matrix may contribute to the selective regulation of those metabolic activities in the cell that have adenine nucleotide dependent steps localized to the mitochondrial compartment (gluconeogenesis, urea synthesis, mitochondrial biogenesis, and even oxidative phosphorylation).     

Effects of atractyloside and palmitoyl coenzyme A on calcium transport in cardiac mitochondria.

Palmitoyl CoA (PCoA) and the adenine translocase inhibitor atractyloside (ATR) appear to produce a similar effect in discharging accumulated calcium from cardiac mitochondria. Although mitochondrial respiration is stimulated upon addition of either PCoA or ATR to preparations preloaded with calcium, the effect is not the same as that produced by classical uncouplers. PCoA and ATR also do not interfere with respiration-supported calcium uptake by mitochondria. The presence of exogenous ATP can prevent the calcium discharging effects of PCoA or ATR. Carnitine will prevent the PCoA calcium discharging effect, but has no effect on ATR-induced discharge. It is suggested the PCoA may act at a site on or near the adenine translocase, perhaps through allosteric interaction, to produce an efflux of calcium from mitochondria. The results also suggest that the internal adenine nucleotide pool plays a significant role in mitochondrial calcium retention.     

Cytochrome c biogenesis in mitochondria--Systems III and V

In c-type cytochromes, heme becomes covalently attached to the polypeptide chain by a reaction between the vinyl groups of the heme and cysteine thiols from the protein. There are two such cytochromes in mitochondria: cytochrome c and cytochrome c(1). The heme attachment is a post-translational modification that is catalysed by different biogenesis proteins in different organisms. Three types of biogenesis system are found or predicted in mitochondria: System I (the cytochrome c maturation system); System III (termed holocytochrome c synthase (HCCS) or heme lyase); and System V. This review focuses primarily on cytochrome c maturation in mitochondria containing HCCS (System III). It describes what is known about the enzymology and substrate specificity of HCCS; the role of HCCS in human disease; import of HCCS into mitochondria; import of apocytochromes c and c(1) into mitochondria and the close relationships with HCCS-dependent heme attachment; and the role of the fungal cytochrome c biogenesis accessory protein Cyc2. System V is also discussed; this is the postulated mitochondrial cytochrome c biogenesis system of trypanosomes and related organisms. No cytochrome c biogenesis proteins have been identified in the genomes of these organisms whose c-type cytochromes also have a unique mode of heme attachment.     

Calcium gradients in tip growing plant cells visualized by chlorotetracycline fluorescence

With chlorotetracycline (CTC)-fluorescence a tip-to-base Ca²⁺ gradient is visualized in all tested, tip-growing plant cells: pollen tubes of Lilium longiflorum, root hairs of Lepidium sativum, moss caulonema of Funaria hygrometrica, fungal hyphae of Achlya and in the alga Acetabularia mediterranea. The fluorescence gradients in the different species vary in intensity and extension. Sometimes a punctate mobile CTC-fluorescence, in the size range of mitochondria, is observed. Bursting cells lose their fluorescence rapidly, indicating a cytoplasmic localization of the gradient. Only in Acetabularia is the wall also fluorescent with CTC. The results are interpreted as evidence for a general role of a calcium gradient in tip growth.Abbreviation CTC chlorotetracycline     

Open-Loop Control of Oxidative Phosphorylation in Skeletal and Cardiac Muscle Mitochondria by Ca2+

In cardiac muscle, mitochondrial ATP synthesis is driven by demand for ATP through feedback from the products of ATP hydrolysis. However, in skeletal muscle at higher workloads there is an apparent contribution of open-loop stimulation of ATP synthesis. Open-loop control is defined as modulation of flux through a biochemical pathway by a moiety, which is not a reactant or a product of the biochemical reactions in the pathway. The role of calcium, which is known to stimulate the activity of mitochondrial dehydrogenases, as an open-loop controller, was investigated in isolated cardiac and skeletal muscle mitochondria. The kinetics of NADH synthesis and respiration, feedback from ATP hydrolysis products, and stimulation by calcium were characterized in isolated mitochondria to test the hypothesis that calcium has a stimulatory role in skeletal muscle mitochondria not apparent in cardiac mitochondria. A range of respiratory states were obtained in cardiac and skeletal muscle mitochondria utilizing physiologically relevant concentrations of pyruvate and malate, and flux of respiration, NAD(P)H fluorescence, and rhodamine 123 fluorescence were measured over a range of extra mitochondrial calcium concentrations. We found that under these conditions calcium stimulates NADH synthesis in skeletal muscle mitochondria but not in cardiac mitochondria.     

Intracellular divalent cation release in pancreatic acinar cells during stimulus-secretion coupling. II. Subcellular localization of the fluorescent probe chlorotetracycline

Subcellular distribution of the divalent cation-sensitive probe chlorotetracycline (CTC) was observed by fluorescence microscopy in isolated pancreatic acinar cells, dissociated hepatocytes, rod photoreceptors, and erythrocytes. In each cell type, areas containing membranes fluoresced intensely while areas containing no membranes (nuclei and zymogen granules) were not fluorescent. Cell compartments packed with rough endoplasmic reticulum or Golgi vesicles (acinar cells) or plasma membrane-derived membranes (rod outer segments) exhibited a uniform fluorescence. In contrast, cell compartments having large numbers of mitochondria (hepatocytes and the rod inner segment) exhibited a punctate fluorescence. Punctate fluorescence was prominent in the perinuclear and peri-granular areas of isolated acinar cells during CTC efflux, suggesting that under these conditions mitochondrial fluorescence may account for a large portion of acinar cell fluorescence. Fluorometry of dissociated pancreatic acini, preloaded with CTC, showed that application of the mitochondrial inhibitors antimycin A, NaCN, rotenone, or C1CCP, or of the divalent cation ionophore A23187 (all agents known to release mitochondrial calcium) rapidly decreased the fluorescence of acini. In the case of mitochondrial inhibitors, this response could be elicited before but not following the loss of CTC fluorescence induced by bethanechol stimulation. Removal of extracellular Ca2+ and Mg2+ or addition of EDTA also decreased fluorescence but did not prevent secretagogues or mitochondrial inhibitors from eliciting a further response. These data suggest that bethanechol acts to decrease CTC fluorescence at the same intracellular site as do mitochondrial inhibitors. This could be due to release of calcium from either mitochondria or another organelle that requires ATP to sequester calcium.     

A perspective on the determination of mitochondrial biogenesis

The measurement of mitochondrial biogenesis is important in the determination of aging and disease processes and the assessment of countermeasurements to them. We argue that the frequently used assessments of cell signaling, mitochondrial protein mRNA, mitochondrial protein expression or enzyme activity, and mitochondrial density are not measurements that can lead to conclusions about mitochondrial biogenesis. Instead, we propose that only measurements of mitochondrial synthesis are indicative of biogenesis. Clarification of this issue will hopefully result in further differentiation of the processes important for morphological and functional changes of mitochondria.     

Oscillatory ROP GTPase activation leads the oscillatory polarized growth of pollen tubes

Oscillation regulates a wide variety of processes ranging from chemotaxis in Dictyostelium through segmentation in vertebrate development to circadian rhythms. Most studies on the molecular mechanisms underlying oscillation have focused on processes requiring a rhythmic change in gene expression, which usually exhibit a periodicity of >10 min. Mechanisms that control oscillation with shorter periods (<10 min), presumably independent of gene expression changes, are poorly understood. Oscillatory pollen tube tip growth provides an excellent model to investigate such mechanisms. It is well established that ROP1, a Rho-like GTPase from plants, plays an essential role in polarized tip growth in pollen tubes. In this article, we demonstrate that tip-localized ROP1 GTPase activity oscillates in the same frequency with growth oscillation, and leads growth both spatially and temporally. Tip growth requires the coordinate action of two ROP1 downstream pathways that promote the accumulation of tip-localized Ca2+ and actin microfilaments (F-actin), respectively. We show that the ROP1 activity oscillates in a similar phase with the apical F-actin but apparently ahead of tip-localized Ca2+. Furthermore, our observations support the hypothesis that the oscillation of tip-localized ROP activity and ROP-dependent tip growth in pollen tubes is modulated by the two temporally coordinated downstream pathways, an early F-actin assembly pathway and a delayed Ca2+ gradient-forming pathway. To our knowledge, our report is the first to demonstrate the oscillation of Rho GTPase signaling, which may be a common mechanism underlying the oscillation of actin-dependent processes such as polar growth, cell movement, and chemotaxis.     

Cellular functions of cardiolipin in yeast

Cardiolipin (CL), the signature lipid of mitochondria, plays a critical role in mitochondrial function and biogenesis. The availability of yeast mutants blocked in CL synthesis has facilitated studies of the biological role of this lipid. Perturbation of CL synthesis leads to growth defects not only during respiratory growth but also under conditions in which respiration is not essential. CL was shown to play a role in mitochondrial protein import, cell wall biogenesis, aging and apoptosis, ceramide synthesis, and translation of electron transport chain components. The genetic disorder Barth syndrome (BTHS) is caused by mutations in the tafazzin gene resulting in decreased total CL levels, accumulation of monolysocardiolipin (MLCL), and decreased unsaturated fatty acyl species of CL. The variation in clinical presentation of BTHS indicates that other physiological factors play a significant role in modifying the phenotype resulting from tafazzin deficiency. Elucidating the functions of CL is expected to shed light on the role of this important lipid in BTHS and other disorders of mitochondrial dysfunction.     

Evolution of macromolecular import pathways in mitochondria,hydrogenosomes and mitosomes

All eukaryotes require mitochondria for survival and growth. The origin of mitochondria can be traced down to a single endosymbiotic event between two probably prokaryotic organisms. Subsequent evolution has left mitochondria a collection of heterogeneous organelle variants. Most of these variants have retained their own genome and translation system. In hydrogenosomes and mitosomes, however, the entire genome was lost. All types of mitochondria import most of their proteome from the cytosol, irrespective of whether they have a genome or not. Moreover, in most eukaryotes, a variable number of tRNAs that are required for mitochondrial translation are also imported. Thus, import of macromolecules, both proteins and tRNA, is essential for mitochondrial biogenesis. Here, we review what is known about the evolutionary history of the two processes using a recently revised eukaryotic phylogeny as a framework. We discuss how the processes of protein import and tRNA import relate to each other in an evolutionary context.     

Conserved features of germination and polarized cell growth: a few insights from a pollen-fern spore comparison

BACKGROUND: The germination of both pollen and fern spores results in the emergence of a cell-pollen tube from pollen, rhizoid from spore-that grows in a polar fashion, primarily at its apical end. In both of these tip-growing cells, the delivery of secretory vesicles to the growing end is guided in part by a calcium gradient, with calcium entering at the tip where it is most highly concentrated. The similarities between the two systems extend beyond tip-focused calcium gradients to encompass signalling pathways and elements including calmodulin, nitric oxide, annexins and Rop-GTPases. SCOPE AND AIMS: This review is limited to those pathways and elements that function similarly in fern and pollen systems based on currently available evidence. The aim is to illustrate the common mechanisms by which tip growth occurs, facilitate further investigations into this area, and examine the implications for the evolutionarily conserved control of tip growth. CONCLUSIONS: The interplay of calcium, nitric oxide and other effectors in both pollen and fern spores suggests certain signalling pathways became important regulators of germination and growth early in the evolution of land plants. Both large- and small-scale comparative genomic methods have shown to be promising in their ability to find new and relevant comparisons for further research. Cross-species comparisons may serve to speed up this process by highlighting both basic pathways and system-specific deviations.