The speed of mitochondrial movement is regulated by the cytoskeleton and myosin in Picea wilsonii pollen tubes.

Strategic control of mitochondrial movements and cellular distribution is essential for correct cell function and survival. However, despite being a vital process, mitochondrial movement in plant cells is a poorly documented phenomenon. To investigate the roles of actin filaments and microtubules on mitochondrial movements, Picea wilsonii pollen tubes were treated with two microtubule-disrupting drugs, two actin-disrupting drugs and a myosin inhibitor. Following these treatments, mitochondrial movements were characterized by multiangle evanescent wave microscopy and laser-scanning confocal microscopy. The results showed that individual mitochondria underwent three classes of linear movement: high-speed movement (instantaneous velocities >5.0 microm/s), low-speed movement (instantaneous velocities <5.0 microm/s) and variable-speed movement (instantaneous velocities ranging from 0.16 to 10.35 microm/s). 10 nM latrunculin B induced fragmentation of actin filaments and completely inhibited mitochondrial vectorial movement. Jasplakinolide treatment induced a 28% reduction in chondriome motility, and dramatically inhibition of high-speed and variable-speed movements. Treatment with 2,3-butanedione 2-monoxime caused a 61% reduction of chondriome motility, and the complete inhibition of high-speed and low-speed movements. In contrast to actin-disrupting drugs, microtubule-disrupting drugs caused mild effects on mitochondrial movement. Taxol increased the speed of mitochondrial movement in cortical cytoplasm. Oryzalin induced curved mitochondrial trajectories with similar velocities as in the control pollen tubes. These results suggest that mitochondrial movement at low speeds in pollen tubes is driven by myosin, while high-speed and variable-speed movements are powered both by actin filament dynamics and myosin. In addition, microtubule dynamics has profound effects on mitochondrial velocity, trajectory and positioning via its role in directing the arrangement of actin filaments.

Actin turnover is required for myosin-dependent mitochondrial movements in Arabidopsis root hairs.

BACKGROUND: Previous studies have shown that plant mitochondrial movements are myosin-based along actin filaments, which undergo continuous turnover by the exchange of actin subunits from existing filaments. Although earlier studies revealed that actin filament dynamics are essential for many functions of the actin cytoskeleton, there are little data connecting actin dynamics and mitochondrial movements. METHODOLOGY/PRINCIPAL FINDINGS: We addressed the role of actin filament dynamics in the control of mitochondrial movements by treating cells with various pharmaceuticals that affect actin filament assembly and disassembly. Confocal microscopy of Arabidopsis thaliana root hairs expressing GFP-FABD2 as an actin filament reporter showed that mitochondrial distribution was in agreement with the arrangement of actin filaments in root hairs at different developmental stages. Analyses of mitochondrial trajectories and instantaneous velocities immediately following pharmacological perturbation of the cytoskeleton using variable-angle evanescent wave microscopy and/or spinning disk confocal microscopy revealed that mitochondrial velocities were regulated by myosin activity and actin filament dynamics. Furthermore, simultaneous visualization of mitochondria and actin filaments suggested that mitochondrial positioning might involve depolymerization of actin filaments on the surface of mitochondria. CONCLUSIONS/SIGNIFICANCE: Base on these results we propose a mechanism for the regulation of mitochondrial speed of movements, positioning, and direction of movements that combines the coordinated activity of myosin and the rate of actin turnover, together with microtubule dynamics, which directs the positioning of actin polymerization events.

Calmodulin binds to extracellular sites on the plasma membrane of plant cells and elicits a rise in intracellular calcium concentration.

Calmodulin (CaM) is a highly conserved intracellular calcium sensor. In plants, CaM also appears to be present in the apoplasm, and application of exogenous CaM has been shown to influence a number of physiological functions as a polypeptide signal; however, the existence and localization of its corresponding apoplasmic binding sites remain controversial. To identify the site(s) of action, a CaM-conjugated quantum dot (QD) system was employed for single molecule level detection at the surface of plant cells. Using this approach, we show that QD-CaM binds selectively to sites on the outer surface of the plasma membrane, which was further confirmed by high resolution transmission electron microscopy. Measurements of Ca(2+) fluxes across the plasma membrane, using ion-selective microelectrodes, demonstrated that exogenous CaM induces a net influx into protoplasts. Consistent with these flux studies, calcium-green-dextran and FRET experiments confirmed that applied CaM/QD-CaM elicited an increase in cytoplasmic Ca(2+) levels. These results support the hypothesis that apoplasmic CaM can act as a signaling agent. These findings are discussed in terms of CaM acting as an apoplasmic peptide ligand to mediate transmembrane signaling in the plant kingdom.

Integrative proteomic and cytological analysis of the effects of extracellular Ca(2+) influx on Pinus bungeana pollen tube development.

Ca (2+) is an essential ion in the control of pollen germination and tube growth. However, the control of pollen tube development by Ca (2+) signaling and its interactions with cytoskeletal components, energy-providing pathways, and cell-expansion machinery remain elusive. Here, we used nifedipine (Nif) to study Ca (2+) functions in differential protein expression and other cellular processes in Pinus bungeana pollen tube growth. Proteomics analysis indicated that 50 proteins showed differential expression with varying doses of Nif. Thirty-four of these were homologous to previously reported proteins and were classified into different functional categories closely related to tip-growth machinery. Blocking the L-type Ca (2+) channel with Nif in the pollen tube membrane induced several early alterations within a short time, including a reduction of extracellular Ca (2+) influx and a subsequently dramatic decrease in cytosolic free Ca (2+) concentration ([Ca (2+)] c), concomitant with ultrastructural abnormalities and changes in the abundance of proteins involved in energy production and signaling. Secondary alterations included actin filament depolymerization, disrupted patterns of endocytosis/exocytosis, and cell wall remodeling, along with changes in the proteins involved in these processes. These results suggested that extracellular Ca (2+) influx was necessary for the maintenance of the typical tip-focused [Ca (2+)] c gradient in the P. bungeana pollen tube, and that reduced adenosine triphosphate production (ATP), depolymerization of the cytoskeleton, and abnormal endocytosis/exocytosis, together with enhanced rigidity of cell walls, were responsible for the growth arrest observed in pollen tubes treated with Nif.