Biol. Bull. 201: 245-246. (October 2001)
© 2001 Marine Biological Laboratory
Microsporidian Spore/Sporoplasm Dynactin in Spraguea
Earl Weidner
Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803
Intracellular protistan parasites have evolved a diversity of adaptations for survival and replication within host cell vacuoles. Some of these adaptations require specific membrane-inserted or surface-attached proteins on the vacuolar envelopes. However, intracellular microsporidian parasites are not in vacuoles; rather, they locate directly in contact with the host cell cytoplasm. This position in the host cytoplasm may be partly due to their means of entry through injection by an invasion tube. Within the microsporidian genus Spraguea, the parasites are confined to neuronal fiber axoplasms in the central nervous system of anglerfish, genus Lophius. The supramedullary neurons are frequently parasitized by Spraguea, and the colonies locate in the proximal regions of fibers adjoining the nerve cell bodies. The supramedullary neurons send fibers to the cutaneous areas in fish (1). Recent studies of puffer fish, genus Takifuga, indicate that these fibers innervate the cutaneous mucous glands (2); this observation is supported by our preliminary investigations on anglerfish. The mucous gland domains of anglerfish skin are the most common sites where infective Spraguea spores activate and discharge their sporoplasms. Mucus is a major activator of Spraguea spores and is a primary factor in effecting spore discharge (3). So the sporoplasms are likely to be introduced directly into the nerve endings surrounding the cutaneous glands in anglerfish. However, established infections of Spraguea are always found at the proximal end of the supramedullary nerve. Our laboratory has therefore hypothesized that Spraguea sporoplasms are equipped with surface proteins that support sporoplasm transport up the fiber to the neuronal cell body area. This study was therefore designed to probe for a dynactin-dynein assemblage because this motor is minus-end-directed and can effect linkups between membrane and microtubules.
To search for dynactin-dynein motor molecules in Spraguea sporoplasms, purified spores were incubated in 0.1 M HEPES buffer (pH 7.0) and transferred to glass coverslips. The spores were activated to discharge their sporoplasms by a method described earlier (3). The discharged sporoplasms retain their attachment to the glass during subsequent washing episodes that remove unfired and discharged spores; the wash solution was 0.1% concanavalin A made up in 0.1 M HEPES (pH 7.0).
The isolated sporoplasms were subjected to optical probes and Western blots using antibodies to dynactin peptides p150glued, p50, Arpl and intermediate dynein chains. The Western blots to Spraguea sporoplasm proteins indicated that, whether whole sporoplasm samples were tested, or only the sporoplasm outer membrane isolates, all four of these peptides were present. Ultrastructural immunolabeling with an antibody-peroxidase probe for p150glued showed the label binding uniformly over the sporoplasm surface (Fig. 1A, B). These results were also supported by immunogold labeling and immunofluorescence (not shown). Since I hypothesized that the dynactin assemblage should be associated with the membrane within unfired spores, tests were made to determine where the dynactin is located in such spores. Spores were prefixed with 1% glutaraldehyde and subsequently partially disrupted by the shearing action of a glass homogenizer. These spores were then subjected to the p150glued antibody probe, and this was visualized with a second antibody coupled to peroxidase. The results showed that the label reacts within the polaroplast domain (Fig. 1C, D). The position of the dynactin within the polaroplast supports an old idea: that the spore discharges a tube through which the membrane of the polaroplast everts to form the sac. The cytoplasm and nucleus are thought to be introduced into the everted polaroplast-derived sac. That the sporoplasm membrane is a secondhand membrane derived from the polaroplast organelle is supported by the absence of cholesterol in the outer envelope (3) and the absence of lectin-binding molecules (unpubl. obs.).
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Figure 1. Spraguea spp. sporoplasm with peroxidase label directly against p150glued. Sporoplasms are exceedingly permeable and thus devoid of some internal matrix; however, the sporoplasm surfaces were consistently labeled with external peroxidase probe (A). (B) This image is a higher magnification of A. (C) The sporoplasm is within an unfired spore and the peroxidase label is within the polaroplast (arrow). (D) This is an enlarged image of C. Bar scales represent 0.25 µm.
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Literature Cited
-
Funakoshi, K., T. Abe, and R. Kishida. 1995. J. Comp. Neurol.,358:552–562.[Medline]
-
Funakoshi, K., T. Kadota, Y. Atobe, M. Nakano, R. Goris, and R. Kishida. 1998. Neurosci. Lett.,258:171–174.[ISI][Medline]
-
Weidner, E., and A. Findley. 1999. Biol. Bull., 197:270–271.
