The actin cytoskeleton has one of the central roles in the physiology of eukaryotic cells and is also involved in the virus-host interactions required for trafficking of viral proteins and particles within and between cells (51
). In plants, the actomyosin motility system functions in cytoplasmic streaming that results in rapid movement of different organelles, including Golgi stacks and peroxisomes (60
). Actin MFs were also implicated in vesicular trafficking and cell plate formation (5
). Furthermore, several studies suggested that actin and myosin may participate in PD biogenesis and function (13
). The data presented in this paper provide experimental evidence for the role of MFs in protein transport to PDs.
Our previous work using electron microscopy demonstrated that Hsp70h is present in PDs of BYV-infected cells (36
). Here we show that Hsp70h localizes to PDs of noninfected cells in the absence of other viral proteins. This conclusion is based on a characteristic appearance of paired Hsp70h-containing granules at opposite sides of cell walls, and colocalization of these granules with an established PD marker, TMV MP-GFP. A very similar pattern of peripheral localization, as well as colocalization with TMV MP-GFP, was observed for a recently discovered cellular PD-residential protein, AtRGP2 (57
). Because the resolution of the light microscope is not sufficient to detect individual PDs, we assume that the observed granules of the nontagged or fluorophore-tagged Hsp70h or TMV MP accumulate in the PD-rich areas or pit fields.
In addition to immobile granules of Hsp70h tightly associated with the plasma membrane and cell wall, time-lapse confocal microscopy revealed granules that moved with a mean speed of ~1 μm/s. We found that these cytoplasmic granules were associated with MFs, but not with microtubules. To identify mechanisms responsible for granule transport, we used seven drugs that affected either microtubular or actomyosin motility system. These experiments demonstrated that drugs that either disassembled MFs or inhibited myosins without affecting MF integrity completely abolished motility of the Hsp70h granules. Moreover, treatment with these drugs prevented formation of the peripheral immobile granules and resulted in a diffuse localization of Hsp70h in the cortical cytoplasm.
The negative effects of the MF- and myosin-specific drugs on both motility and localization of the Hsp70h do not necessarily imply that the former is required for the latter. However, the most parsimonious hypothesis states that trafficking of Hsp70h in association with MFs is the pathway for transporting this viral protein to the PD. Tight association of the cytoplasmic Hsp70h granules with MFs is in accord with this hypothesis. Furthermore, this association strongly suggests that Hsp70h translocation involves specific interaction with the actomyosin motility system rather than passive trafficking due to cytoplasmic streaming.
In accord with previous research, microtubule-specific drugs had only a moderate negative effect on cytoplasmic streaming (43
). These drugs did not significantly inhibit trafficking, or altered partitioning of the Hsp70h to the cell periphery. These results indicated that the intact microtubular cytoskeleton is not required for Hsp70h localization to pit fields.
Interestingly, none of the tested inhibitors was able to markedly affect PD targeting of the GFP-tagged TMV MP. These unexpected observations could be due to higher tolerance of this process to drug treatments. An alternative explanation implies a cytoskeleton-independent mechanism of TMV MP targeting and therefore suggests that more than one mechanism could be used by viral proteins to reach PDs. The ability of TMV MP to interact with the cell-wall-associated pectin methylesterase could be a part of such alternative mechanism (9
). It should also be emphasized that our experiments with TMV MP indicate that the drug treatments do not disrupt PDs that remain competent for TMV MP docking.
It seems likely that trafficking of the viral proteins, genomes, and particles within and between cells relies on preexisting pathways of intercellular communication used by host NCAPs (31
). It will be interesting to see if the actin cytoskeleton is involved in the targeting of NCAPs to PDs. Recent work showed that the non-cell-autonomous behavior of certain plant Hsc70s is mediated by a specific amino acid sequence motif (3
). In the case of another NCAP, transcription factor KN1, such behavior relies on a C-terminal homeodomain (25
). None of these signals is present in BYV Hsp70h. Our previous work showed that the GFP fusions of either the N-terminal ATPase domain or C-terminal domain of Hsp70h localized to peripheral punctate bodies (53
). These results suggested that Hsp70h may possess redundant signals for peripheral targeting.
The pathways of MP translocation upon transient expression do not necessarily reproduce events of the viral infection that may involve association of MPs with other viral proteins, genomes, or virions. Indeed, it has been demonstrated that TMV
MP colocalizes and traffics with viral replication complexes (23
). The TGB MPs are involved in multiple interactions between themselves (18
), while tubule-forming MPs enclose virions into the tubes formed during infection (29
). In addition to accumulation in PDs, BYV Hsp70h is incorporated to virion tails that function in virus transport (2
We have also tested effects of cytoskeletal inhibitors on the BYV replication and cell-to-cell movement. It was revealed that while intact microtubules are dispensable for both processes, MF disassembly by drugs dramatically reduces viral RNA accumulation and abolishes BYV movement from cell to cell (A. I. Prokhnevsky and V. V. Dolja, unpublished data). Therefore, demonstration of a possible direct function of MFs in BYV movement that might be overshadowed in these experiments by MF requirement for replication needs to be addressed using an alternative approach.
Investigation of the targeting pathways of individual viral proteins described in this and other recent papers (18
) continues to provide important insight into molecular mechanisms of virus interactions with the plant cells. In addition to findings with NCAP trafficking in plants, one of the herpesvirus tegument proteins exhibits NCAP-like behavior in being able to translocate viral mRNAs between animal cells (15
). Moreover, cultured animal cells can interconnect via nanotubules that facilitate intercellular vesicular trafficking in an actomyosin-dependent manner (56
). It appears that direct intercellular communications involving actin cytoskeleton may become a common theme in plant and animal cell biology and virology.