Actin Purified from Maize Pollen Functions in Living Plant Cells.

Abstract

A vast array of actin binding proteins (ABPs), together with intracellular signaling molecules, modulates the spatiotemporal distribution of actin filaments in eukaryotic cells. To investigate the complex regulation of actin organization in plant cells, we designed experiments to reconstitute actin-ABP interactions in vitro with purified components. Because vertebrate skeletal a-actin has distinct and unpredictable binding affinity for nonvertebrate ABPs, it is essential that these in vitro studies be performed with purified plant actin. Here, we report the development of a new method for isolating functional actin from maize pollen. The addition of large amounts of recombinant profilin to pollen extracts facilitated the depolymerization of actin filaments and the formation of a profilin-actin complex. The profilin-actin complex was then isolated by affinity chromatography on poly-L-proline-Sepharose, and actin was selectively eluted with a salt wash. Pollen actin was further purified by one cycle of polymerization and depolymerization. The recovery of functional actin by this rapid and convenient procedure was substantial; the average yield was 6 mg of actin from 10 g of pollen. We undertook an initial physicochemical characterization of this native pollen actin. Under physiological conditions, pollen actin polymerized with kinetics similar in quality to those for vertebrate a-actin and had a critical concentration for assembly of 0.6 pM. Moreover, pollen actin interacted specifically and in a characteristic fashion with several ABPs. Tradescantia cells were microinjected and used as an experimental system to study the behavior of pollen actin in vivo. We demonstrated that purified pollen actin ameliorated the effects of injecting excess profilin into live stamen hair cells.

Incorporation of mammalian actin into microfilaments in plant cell nucleus

“…This observation is consistent with the earlier findings that fluorescence analogue cytochemistry with mammalian actin does not work in plant cells [9,10]. Also, it puts under doubt the functionality of the chimeric rabbit-plant microfilaments.…”

“…Previous attemps to apply analogue cytochemistry with fluorescently labeled mammalian actin in plant cells have failed because rabbit muscle actin appeared to poison living plant cells [9,10] whereas maize pollen actin have worked perfectly at the same needle concentration. In the current work, actin-Alexa Fluor ® 488 conjugate was used because it has significantly better photostability and brighter fluorescence than classical fluorescein or rhodamine derivates.…”

“…In the current work, actin-Alexa Fluor ® 488 conjugate was used because it has significantly better photostability and brighter fluorescence than classical fluorescein or rhodamine derivates. Application of this conjugate made possible to observe the injected actin keeping the needle concentration as low as 5 μM (that is 13 times lower than 65 μM [10]) to avoid potential toxic effects. Also, the injection volume was kept to minimum using manual oil injector.…”

“…Mammalian fluorescently labeled neurotubulin has been shown to incorporate into plant microtubules [5], but fluorescent staining of plant microfilaments in living cells has been carried out indirectly, using phalloidin derivates [6], GFP-mouse talin fusion protein [7], and fluorescently labeled fimbrin [8]. The ability of heterologous fluorescent actin to incorporate into endogenous pool of plant microfilaments has been under doubt [9,10] but the reason why mammalian actin did not incorporate into plant microfilaments during few published experiments is still unclear [10,11]. In the current work we applied the commercially available rabbit actin conjugated with Alexa Fluor ® , a dye with long fluorescence lifetime as a tool for visualization of microfilaments in living cells.…”

Contractile Proteins in Chemical Signal Transduction in Plant Microspores

“…Pollen germination indicated by the appearance and growth of the pollen tube depends on the activity of contractile proteins actin and myosin (Ren et al ., 1997;Taylor and Hepler, 1997;Cai et al ., 2000;Chen C.Y. Pollen germination indicated by the appearance and growth of the pollen tube depends on the activity of contractile proteins actin and myosin (Ren et al ., 1997;Taylor and Hepler, 1997;Cai et al ., 2000;Chen C.Y.…”