Actin polymerization is essential for pollen tube growth.

Actin microfilaments, which are prominent in pollen tubes, have been implicated in the growth process; however, their mechanism of action is not well understood. In the present work we have used profilin and DNAse I injections, as well as latrunculin B and cytochalasin D treatments, under quantitatively controlled conditions, to perturb actin microfilament structure and assembly in an attempt to answer this question. We found that a approximately 50% increase in the total profilin pool was necessary to half-maximally inhibit pollen tube growth, whereas a approximately 100% increase was necessary for half-maximal inhibition of cytoplasmic streaming. DNAse I showed a similar inhibitory activity but with a threefold more pronounced effect on growth than streaming. Latrunculin B, at only 1--4 nM in the growth medium, has a similar proportion of inhibition of growth over streaming to that of profilin. The fact that tip growth is more sensitive than streaming to the inhibitory substances and that there is no correlation between streaming and growth rates suggests that tip growth requires actin assembly in a process independent of cytoplasmic streaming.

Free calcium in Micrasterias: local gradients are not detected in growing lobes.

Intracellular free Ca2+ ([Ca2+]) has been measured in growing unicells of two species of the green alga, Micrasterias, which have been injected with the indicator dye fura-2-dextran. Ratiometric imaging of Micrasterias denticulata yields levels of 170 to 200 nM [Ca2+] but fails to reveal a significant [Ca2+] gradient associated with the tips of growing lobes, or in any other region of the cell. In Micrasterias muricata slight elevations from a basal value of 350 to 500 nM have been observed, but these might be due to a general inward leakage of Ca2+ at the plasma membrane which is enhanced at the narrow lobes of this cell because of their greater relative surface to volume ratio. Experimental perturbation of the intracellular [Ca2+] with injection of the ion or the addition of the non-fluorescent ionophore, Br-A23187, reveal that [Ca2+] elevations can be generated and indicate that if they naturally occurred, the image system would have detected them. Further evidence that [Ca2+] gradients are lacking derives from studies with BAPTA-type buffers. Injection of 5,5'-dibromo BAPTA and 4,4'-difluoro BAPTA, which in several other systems are the most effective at dissipating intracellular [Ca2+] gradients, have no effect on development of Micrasterias. Taken together, these studies indicate that lobe outgrowth in Micrasterias does not occur in association with marked localized [Ca2+] gradients.

Actin microfilaments are associated with the migrating nucleus and the cell cortex in the green alga Micrasterias. Studies on living cells.

Rhodamine-phalloidin or FITC-phalloidin has been injected in small amounts into living, developing cells of Micrasterias denticulata and the stained microfilaments visualized by confocal laser scanning microscopy. The results reveal that two different actin filament systems are present in a growing cell: a cortical actin network that covers the inner surface of the cell and is extended far into the tips of the lobes in both the growing and the nongrowing semicell; it is also associated with the surface of the chloroplast. The second actin system ensheathes the nucleus at the isthmus-facing side during nuclear migration. Its arrangement corresponds to that of the microtubule system that has been described in earlier electron microscopic investigations. The spatial correspondence between the distribution of actin filaments and microtubules suggests a cooperation between both cytoskeleton elements in generating the motive force for nuclear migration. The function of the cortical actin network is not yet clear. It may be involved in processes like transport and fusion of secretory vesicles and may also function in shaping and anchoring the chloroplast.