The Cytoskeleton and Its Regulation by Calcium and Protons

Abstract

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.

Figure 1.

Lily pollen tubes, which have been treated with different reporters or stains, show the distribution of free calcium (A), pH (B), F-actin (C), and the actin-binding protein ADF (D). A, Microinjection of the calcium-sensitive dye Fura-2-dextran allows one to image the distribution of free calcium in a living lily pollen tube. Ratiometric analysis reveals that the calcium is maximal at the extreme apex, where the concentrations are 1 to 10 µm. Back from the tip, the calcium concentration drops sharply, reaching the basal level of 0.15 µm within 15 to 20 µm. From Rounds et al. (2011). B, In this instance, the living pollen tube has been microinjected with BCECF-dextran, a pH-sensitive dye. The ratiometric image reveals a slightly acidic region at the extreme apex followed by a prominent alkaline band (orange/red; pH 7.5), starting a few micrometers behind the tip and extending rearward for an additional 10 to 20 µm. Thereafter, the pH approaches neutrality. From Rounds et al. (2011). C, In this image, the lily pollen tube has been preserved through rapid freeze fixation, followed by rehydration and staining with an anti-actin antibody. The resulting confocal fluorescence image reveals the striking cortical actin fringe that begins a few micrometers back from the tip and extends rearward for 5 to 10 µm. The MFs in the fringe are organized as a palisade in which the individual elements are aligned parallel to the long axis of the pollen tube. Behind the fringe, the MFs are also longitudinally oriented, but they appear to be much less dense than in the fringe. From Lovy-Wheeler et al. (2005). D, In this example, a lily pollen tube, which has been preserved through rapid freeze fixation and rehydration, has been stained with an antibody to lily ADF1. The stained region starts a few micrometers back from the apex and extends rearward 3 to 5 µm. By comparison with C, it is obvious that ADF colocalizes with the actin fringe, perhaps especially the forward edge of the fringe. Also note that ADF (D) and the actin fringe (C) colocalize with the alkaline band (B). From Lovy-Wheeler et al. (2006). Bar, 10 μm.

Figure 2.

A, This image shows an electron micrograph of a dividing barley leaf mesophyll cell in metaphase that has been fixed in glutaraldehyde and postfixed in osmium tetroxide plus potassium ferricyanide. The osmium/ferricyanide treatment markedly contrasts the ER and reveals that, while the nuclear envelope has broken down, the mitotic spindle remains surrounded by ER. The image further shows that ER accumulates at the spindle poles and that some elements extend into the spindle interior, usually along kinetochore MTs. Bar = 1 µm. From Hepler (1980). B and C, At higher magnification than in A, these images show that tubular elements of ER extend along the full length of kinetochore MTs. In B, the ER has been contrasted by postfixation with osmium/ferricyanide, whereas in C, only standard glutaraldehyde/osmium fixation has been used. Both images show the close apposition of interpenetrating ER with kinetochore MTs. C also shows some dictyosome vesicles (D). Bars = 0.5 µm. From Hepler (1980).

Figure 3.

Iontophoretic injection of controlled amounts of calcium during early anaphase produces a clear effect on chromosome motion. In A and B, the injection needle was inserted into the mid plane of the cell, whereas in C, it was inserted into the spindle pole. The microinjection needle contained 20 mm CaCl₂ and 100 mm KCl. The magnitude of the current was then varied in order to produce the desired level of calcium. A, Application of positive current of 1 nA for 10 s, which produces an increase in calcium to approximately 1 µm, causes the chromosomes to increase their rate of motion from 1.1 to 2.1 µm min⁻¹ for approximately 20 s. B, However, application of 2 nA for 10 s causes a brief slowing of chromosome motion. C, Injection of calcium at 2 nA for 10 s to one of the spindle poles causes a brief slowing of chromosomes to the proximal pole while simultaneously accelerating motion to the distal pole. From Zhang et al. (1990b).