Concomitant alterations in distribution of 70 kDa heat shock proteins, cytoskeleton and organelles in heat shocked 9L cells.

Maintenance of cell architecture and positioning of organelles are major functions of the cytoskeleton. On the other hand, induction of heat shock proteins (HSPs) and reorganization of the cytoskeleton are the most significant changes in heat-shocked mammalian cells. We examine the alterations in HSP70 and its constitutively expressed cognate, HSC70, as well as the cytoskeleton and organelles in 9L rat brain tumor cells upon heat shock. We employed fluorescence microscopy and scanning electron microscopy to follow these changes. Levels of HSP70s were quantified by Western blotting. Accumulation of HSC70 was more transient and the protein translocated to and subsequently exited from the nucleus more rapidly than HSP70. Changes in actin microfilaments include the nuclear localization of actin fraction and disappearance of cytoplasmic microfilament bundles, while the cortical actin microfilaments were almost unaffected. Furthermore, microtubules retracted slightly from the cell periphery but remained largely unchanged. In contrast, the intermediate filaments collapsed into the perinuclear region. The mitochondria converted from filamentous into granular forms and clustered in a region overlapping with the collapsed intermediate filaments. All of the above alterations are reversible and largely reverted after 8 h of recovery. The effect on Golgi organization was very transient and the apparatus assumed a normal appearance within 4 h after the heat treatment. The ER, on the other hand, was totally unaffected by the heat treatment. These observations help correlate the sequential events following a stress like heat shock and suggest possible physiological functions of these essential constituents of a cell under stress.

Protocerebral neurons inhibiting proliferation of corpus allatum cells in the cockroach, Diploptera punctata.

A mitotic wave before an increase in juvenile hormone (JH) synthesis was observed in the corpora allata during each ovarian cycle in the cockroach Diploptera punctata and was shown subsequently to be inhibited by the brain until adult females mated. Each corpus allatum (CA) was innervated by groups of neurons in the contralateral pars intercerebralis (PI) and the ipsilateral pars lateralis (PL). In this article, a third set of neurons is identified that innervates the CA located extralaterally in the contralateral PL. The topography of the brain neurons innervating the CA was reconstructed from confocal optical sections after vital staining with two fluorescent carbocyanine dyes, 1,1'-dihexadecyl-3, 3,3',3'-tetramethylindocarbocyanine perchlorate [DiIC(16) (3)] and 3, 3'-dioctadecyloxacarbocyanine perchlorate [DiOC(18) (3)]. This provided information necessary to disconnect specific pathways within the brain of virgin females and to determine the effect on mitosis in the corpora allata and on the growth of oocytes. Disconnection of the CA from the PI neurons was followed by a prolonged period of moderate mitotic activity in the glands. Disconnection of the CA from PI and contralateral PL neurons was followed by a rapid wave of high mitotic activity in the CA similar to that observed after mating. The ipsilateral PL neurons did not appear to influence CA cell proliferation, because mitotic activity in glands was similar whether or not glands were connected to these neurons. Disconnection of the contralateral PI and/or ipsilateral PL neurons, but not the contralateral PL neurons, from the CA resulted in oocyte growth indicative of increased JH synthesis. The authors conclude that, whereas contralateral PI neurons inhibit both CA cell proliferation and JH synthesis, ipsilateral PL neurons affect only JH synthesis, and contralateral PL neurons modulate cell proliferation slightly only.

Three-dimensional mapping of brain neuropils in the cockroach, Diploptera punctata.

Herein, we present a complete three-dimensional (3D) map of major neuropil structures in the central brain of the cockroach Diploptera punctata. The positions of the structures have been ascertained by confocal microscopy, which, until now-for reasons of tissue opacity and nonhomogeneity-has been thought impractical in imaging fluorescently labeled structures thicker than 150 microm. In this report, however, we have used digestive enzymes and microwave-aided fixation to stain, clear, and optically section, in its entirety, an intact central brain more than 500 microm thick. The central brain from an adult female cockroach was stained thoroughly with the membrane probe NBD-ceramide and the DNA probe propidium iodide. The central brain as well as such neuropil regions as mushroom bodies, central complex, antennal glomeruli, and lobus glomerulati were individually outlined, segmented, and reconstructed in three dimensions to a spatial resolution of approximately 1 microm in the X-Y plane and 3 microm in the Z plane. The volume and surface area of each neuropil compartment were determined, and Kenyon cells of the mushroom bodies were counted. We determined that each brain hemisphere contains about 230,000 Kenyon cells, 99 antennal lobe glomeruli, and 40 lobus glomerulatus glomeruli. Segmented compartments were assigned as separate channels and merged into a single data base to reconstruct a 3D central brain containing eight different channels. This is the first 3D map at submicron resolution of an entire animal's brain that measures more than 500 microm in thickness.

Morphometric analysis of corpus allatum cells in adult females of three cockroach species.

The number of cells and their sizes in the corpus allatum (CA) of adult female Blattella germanica, Supella longipalpa and Diploptera punctata were determined during oocyte maturation. Cell number and size were directly measured in cell suspensions following enzymatic dissociation of freshly excised CA. Cell numbers were verified by total cell counts in whole-mount CA monolayers and by hemocytometric sampling. In all three species, cell number did not change during the period of CA activation, averaging ca. 2000 cells per gland in B. germanica, 3500 cells per gland in S. longipalpa and 11,000 cells per gland in D. punctata. Cell diameter increased significantly in all three species during this period from a mean value of 8.9 microns to 11.7 microns in B. germanica, from 9.2 microns to 14.6 microns in S. longipalpa and from 10.0 microns to 15.6 microns in D. punctata. During a 4 h incubation period, dissociated CA cells incorporated L-[methyl-3H]-methionine into juvenile hormone-III at rates comparable to intact glands. These data suggest that CA activation in the first ovarian cycle of these species is associated mainly with an increase in cell size with minor changes in cell number.

Neural and hormonal regulation of growth of corpora allata in the cockroach, Diploptera punctata.

DNA synthesis and mitosis in the corpora allata (CA) of adult Diploptera punctata males were investigated with total cell count after 5'-bromo-2'-deoxyuridine immunodetection and colchicine arrestment both in vivo and in vitro. The CA exhibited a single wave of DNA synthesis followed by cell division during the first 4 days after the imaginal ecdysis. A second mitotic wave was experimentally induced after the nervous connections between the CA and the brain were severed on day 4. Spontaneous mitosis was abolished in cockroaches treated with a juvenile hormone (JH) analog. This inhibitory regulation in vivo appeared to act through brain neurosecretory cells since in the denervated CA mitotic activity was unaffected by JH treatment. An in vitro system supporting growth of the corpus allatum was established to study direct hormonal effects. By using continuous bromodeoxyuridine labeling in vitro for 6 days, we showed that DNA synthesis of corpus allatum cells was unaffected by direct contact with JH. In contrast, 20-hydroxyecdysone exerted direct mitogenic action on allatal cells. These and previous results suggest that CA cells alternate between JH synthesis and a proliferative state in which they divide in a self-renewing fashion to yield differentiated progeny. We propose that in newly enclosed adult Diploptera punctata males, low JH titer and high ecdysteroid titer promote mitosis in CA cells. As the ecdysteroid titer declines, JH produced by the CA acts on brain neurosecretory cells which dispatch inhibitory signals through nerves to prevent continuous proliferation of CA cells.

Developmental regulation of juvenile hormone synthesis: ovarian synchronization of volumetric changes of corpus allatum cells in cockroaches.

The corpus allatum (CA) cells of adult Blattella germanica females undergo cyclic volumetric changes in relation to juvenile hormone (JH) synthesis. In intact females the size of CA cells changes synchronously during the gonotrophic cycle, resulting in cyclic JH synthesis. In ovariectomized females volumetric changes among CA cells become asynchronous, resulting in highly variable but high rates of JH synthesis. Injection of the steroid hormone 20-hydroxyecdysone into ovariectomized females with active CA resulted in a transient decline followed by an increase in both CA volume and JH biosynthesis. This response was due to a change in the size distribution of CA cells and not in the total number of CA cells. In ovariectomized females, CA cells can be re-synchronized into a uniform population of small inactive cells with injection of 20-hydroxyecdysone and implantation of an artificial egg-case, mimicking the successive events of ovulation, oviposition and pregnancy.

Glutamate-induced rise in cytosolic calcium concentration stimulates in vitro rates of juvenile hormone biosynthesis in corpus allatum of Diploptera punctata.

We show that in a cockroach, Diploptera punctata, endocrine function of the corpus allatum may be modulated by L-glutamate, a major fast excitatory neurotransmitter in the central nervous system of vertebrates and invertebrates. The widely accepted concept that synthesis of juvenile hormone (JH) depends upon intracellular calcium concentration, is extended by the finding that 60 and 100 microM L-glutamate induces both an increase in calcium concentration in the cytosol of corpus allatum cells, and stimulates JH synthesis in vitro. We show that L-glutamate stimulates JH synthesis by inducing calcium influx since in calcium-free medium the stimulatory effect is not observed. Furthermore, the non-specific glutamate-receptor antagonist, 100 microM kynurenate, and 1.8 mM magnesium, inhibit the stimulatory effect of L-glutamate on JH synthesis in vitro. These results suggest that functional ionotropic glutamate receptors are present on the surface of the cells in corpus allatum, and that rates of JH are at least in part regulated via these receptors.

Nadph-diaphorase activity in corpus allatum cells of the cockroach, Diploptera punctata.

Using the fixation insensitive NADPH-diaphorase reaction as a histochemical marker for the enzyme nitric oxide synthase (NOS), we investigated the possible sites of putatively NOS-related NADPH-diaphorase in the brain and retrocerebral complex of the cockroach, Diploptera punctata. In the cerebral ganglion, NADPH-diaphorase expression was localized in antennal lobes, optic lobes, mushroom bodies and neurosecretory cells. The highest NADPH activity was detected in the corpora allata (CA). Spectrophotometric quantitation indicated that NADPH-diaphorase activity first increased and then decreased (cycled) in the CA of mated females. In addition, during the first ovarian cycle, NADPH-diaphorase activity fluctuated concurrently with cyclic changes in the size of corpus allatum cells. In virgin females, NADPH-diaphorase activity remained at a low level, but it increased if the neural connectives between CA and brain were severed, indicating that the brain inhibited NADPH-diaphorase expression in the CA. Although nerve terminals were abundant in the CA, NADPH-diaphorase was clearly endogenous and synthesized by glandular cells, as was shown by histochemical staining of the cytosol in all dissociated cells of the CA. We have also demonstrated NADPH-diaphorase activity in the CA of the American cockroach Periplaneta americana, the house cricket Acheta domesticus, the lepidopteran Leucania loreyi, and the fruit fly Drosophila melanogaster, suggesting that NOS occurs in the CA of most, if not all insects. It is therefore possible that corpus allatum cells release NO, along with juvenile hormone, which presumably can function as a messenger molecule.

Improved conditions for culture of biosynthetically active cockroach corpora allata.

Currently, short-term culture of insect corpora allata is most often performed in TC199. We now show that L-15B, a medium widely used in arthropod tissue culture, is superior to TC199 for both short- and long-term culture of cockroach corpora allata. In 3-h and 48-h incubations, juvenile hormone biosynthesis by corpora allata from Diploptera punctata was significantly higher in L-15B than in TC199. In addition, in both media, corpora allata activity was significantly improved by flotation of glands at the medium surface. Characteristics of L-15B responsible for its superiority were examined by comparison of gland activities in several TC199 formulations that had been modified in different ways to be more similar to L-15B. Adjusting the osmotic pressure of TC199 (288 mOsm/l) to near that of L-15B (362 mOsm/l) and D. punctata hemolymph (360 mOsm/l) significantly improved gland activity during the second 12 h of a 36-h incubation. Increasing the concentrations of amino acids, sugars, and organic acids in TC199 to the same levels as in L-15B significantly improved gland activity during both the second and third 12-h intervals of a 36-h incubation. These results suggest that L-15B is superior to TC199 because L-15B is isoosmotic with D. punctata hemolymph and because L-15B, like cockroach hemolymph, contains a high level of organic constituents. It is therefore more appropriate to use L-15B than TC199 for short-term in vitro assays of juvenile hormone biosynthesis and for extended corpora allata culture.

In vitro growth of corpora allata from Diploptera punctata.

An in vitro organ culture system was established to support growth of corpora allata from the cockroach Diploptera punctata. During a 1-wk incubation in L-15B medium supplemented with 10% fetal bovine serum (FBS) and 10% cockroach hemolymph, adult male corpora allata exhibited a cycle of de novo DNA synthesis followed by cell division. The number of S-phase cells and metaphase cells per corpus allatum were counted from whole-mount monolayers after labeling in vitro with 5'-bromo-2'-deoxyuridine and exposure to colchicine, respectively. While both FBS and cockroach hemolymph were essential for proliferation of allatal cells, the growth-promoting effect of insect hemolymph was not species-specific and adult female hemolymph was more potent than hemolymph from adult males. Furthermore, DNA synthesis of corpus allatum cells was stimulated in vitro by 20-hydroxyecdysone. This sensitive assay system will be of immense utility in the search for allatal growth factors.

Ultrastructural changes in corpora allata during a cycle of juvenile hormone biosynthesis in embryos of the viviparous cockroach, Diploptera punctata.

We have observed changes with time in the fine structure of corpora allata (CA) during a known cycle of increasing and decreasing juvenile hormone (JH) synthesis in late embryos of Diploptera punctata. A previous report showed that rates of JH release were relatively low in 28-day-old embryos, but CA activity subsequently rose linearly to a peak on about day 42, and thereafter steadily declined to a low level on day 64 just before birth (Holbrook et al., 1997). We now show that, regardless of rate of JH synthesis, CA cells are large and replete with organelles which nevertheless exhibit variable morphology in embryos of different age. Highly active CA cells on day 40 contain abundant ring-form mitochondria, whereas CA cells of low activity on days 28 and 64 contain mitochondria that are rod-shaped or globular. Mitochondrial cristae were scarce and indistinct on day 28 but numerous and well developed on day 64. Endoplasmic reticula (ER) are rare on day 28 and appear in increasing numbers when CA activity rises. On day 40, ER are abundant and often exhibit a whorl-like appearance which is not observed on day 28. After day 44, when biosynthetic activity is declining, whorls of ER gradually decrease in number and are ultimately replaced by vesicular smooth ER on day 64. Neurosecretions are found only after day 38, by which time rates of JH synthesis have increased substantially from those of day 28. Except for membranous autophagic vacuoles, which are frequently found when ER whorls disintegrate as rates of JH synthesis decline toward birth, most autophagic vesicles such as multivesicular vesicles and dense bodies occur only sporadically among CA cells at all examined ages. We conclude that synchronous autophagy of exhausted organelles, which results in atrophy of CA cells and long-term arrest of JH synthesis in adult females of D. punctata, does not occur in embryos. The slow cyclic change in rate of JH synthesis in embryonic CA is most likely due to asynchronous autophagic activity and to alterations in certain unique features of intracellular organelles.

Optical clearing improves the imaging depth and signal-to-noise ratio for digital analysis and three-dimensional projection of the human enteric nervous system.

BACKGROUND: Due to the dispersed nature of neurites and fibers, the microtome-based 2-dimensional histology provides only a limited perspective of the enteric nervous system. To visualize the enteric plexus, we applied optical clearing to avoid scattering in the human ileum to facilitate photon penetration for 3-dimensional (3-D) microscopy of the neural tissue. METHODS: Human ileal specimens were derived by trimming the donor bowel due to its excess length during the clinical trial of small intestinal transplantation. The pan-neuronal marker PGP9.5 was used as the immunostaining target to reveal the enteric plexuses. The labeled tissues were immersed in the optical-clearing solution prior to deep-tissue confocal microscopy. The serial sections were digitally analyzed and processed by reconstruction algorithms for 3-D visualization. KEY RESULTS: Optical clearing of the ileal specimen led to less fluorescence signal decay along the focal path in the tissue and a higher signal-to-noise ratio of the confocal micrographs in comparison with the untreated saline control. Taking advantage of the high signal-to-noise ratio images, we applied software-based signal analysis to identify the presence of the nerve fibers and quantify the signal peaks. The image stacks derived from the serial anatomic micrographs created panoramic views of the gut wall innervations with their associated microstructures. CONCLUSIONS & INFERENCES: We provide an optical approach to improve the imaging depth in 3-D neurohistology of the human ileum. This methodology has significant promise in facilitating our understanding of the enteric nervous system in health and disease.

Formation of a whorl-like autophagosome by Golgi apparatus engulfing a ribosome-containing vacuole in corpora allata of the cockroach Diploptera punctata.

Previously, we showed that synchronous autophagy occurred in the corpora allata during the declining phase of juvenile hormone synthesis in mated adult females of the cockroach Diploptera punctata. At the onset of this process, conspicuous whorl-like autophagosomes were often observed. Each consisted of a ribosome-containing vacuole at the center surrounded by about eight layers of double membranes. Labeling the cis-Golgi cisternae with hot osmium and histochemical detection of thiamine pyrophosphatase in the trans-Golgi cisternae were used to trace the autophagic process. We conclude that the membranes surrounding the whorl-like autophagosome are derived from the entire Golgi apparatus with its cis-cisternae forming the outermost layer and the trans-cisternae the innermost layer. The engulfing process is initiated through direct contact between trans-cisternae of the Golgi apparatus and the isolation membrane of the ribosome-containing vacuole. Formation of a whorl-like autophagosome appears to be a highly regulated process specific to ribosome-containing vacuoles since it occurs only on day 5 and the Golgi apparatus does not recognize membranes of other types of autophagic vesicles or of functional organelles. This novel behavior of the Golgi apparatus in the corpora allata should play an important role in the long-term inhibition of juvenile hormone synthesis (e.g., during gestation) since it results in the depletion of cellular machinery for juvenile hormone synthesis.

Hormonal control of sexual receptivity in cockroaches.

Many animals exhibit specific behaviors associated with sexual receptivity only when they are reproductively competent. In insects with gonadal maturation cycles, these behaviors usually coincide with ovarian maturation. In the cockroach Blattella germanica, juvenile hormone (JH), produced by the corpora allata (CA), regulates female reproductive physiology. Various experimental manipulations, including ablation of the CA, therapy with JH analogs, CA denervation, ovariectomy, and changing nutrient quality, coupled with time-lapse video recording, support the hypothesis that JH also controls female sexual receptivity. A re-examination of the role of the CA in the maturation of male sexual readiness shows that, while sexual behavior develops in the absence of JH in both B. germanica and Supella longipalpa, JH accelerates the expression of sexual readiness.

Arthropod immune system: I. Comparative light and electron microscopic accounts of immunocytes and other hemocytes of Blattella germanica (Dictyoptera: Blattellidae).

Light microscopy (LM) and scanning (SEM) and transmission electron microscopy (TEM) disclose seven types of hemocytes in adults of the German cockroach, Blattella germanica (Dictyoptera: Blattellidae)-namely, prohemocytes (PR), granulocytes (GR), plasmatocytes (PL), spherulocytes (SP), adipohemocytes (AD), coagulocytes (CO), and oenocytoids (OE). Their shape, size, surface appearance, and internal ultrastructure are characterized here. The shapes and relative sizes of hemocytes noted in vitro have been compared with those flowing through the wing veins (in vivo). The GRs constitute more than 90% of the hemocyte population and have roles in the defense system such as encapsulation and phagocytosis. They are characterized by numerous cytoplasmic granules and marginal microtubular bundles, which are commonly noted in the blood cells of both vertebrates and invertebrates, but have been rarely reported in insect hemocytes. The PRs are characterized by being spherical and small, having a relatively large nucleus and a small area of cytoplasm. The polymorphic PLs have some micropapillae on the cell surface and fewer granules than the GRs. The SPs are characterized by an oblate shape, raspberrylike appearance in SEM, and many spherules in the cytoplasm. The ADs are spherical and have large, refringent fat droplets. The COs possess a distinctive cartwheellike appearance when observed by LM but resemble GRs in TEM preparations. The OEs are opaque and often have an eccentric nucleus. Of the seven types of hemocytes, all, except the COs, can be recognized both in vivo and in vitro by LM; the COs can be recognized only in vitro. SEM does not allow the COs to be distinguished from the GRs, except in samples in which coagulation has occurred. All hemocyte types can be recognized by TEM. Thus, all three types of microscopy allow the various types of hemocytes to be recognized; unequivocal discrimination among the cells is best achieved by combining all the three kinds of microscopy.

The role of 20-hydroxyecdysone in stimulating DNA synthesis in corpora allata of the silkworm, Bombyx mori.

Temporal changes in DNA synthesis were examined in the corpora allata of the silkworm Bombyx mori during the penultimate larval stadium. 5'-Bromo-2'-deoxyuridine (BrdU) labeling revealed that allatal DNA synthesis rose early in the stadium, peaking at about 2% of corpus allatum (CA) cells 24 hr after ecdysis. This corresponded to a rising phase of the ecdysteroid titer reported previously. DNA synthesis declined to a nearly undetectable level 1 day later. In larvae fed on leaves coated with 20-hydroxyecdysone (20E), the number of BrdU-labeled cells increased continuously after the normal peak and reached about 8% of CA cells at the end of the stadium. However, total count of CA cells from whole-mount monolayers and colchicine treatment indicated that, regardless of DNA content, CA cells did not divide. To determine if the rise in 20E was responsible for the onset of allatal DNA synthesis, we have established an organ culture system to support de novo DNA synthesis in vitro. The number of BrdU-labeled cells increased with increasing 20E concentration between 0.1 and 0.5 microg ml-1 in the medium, corresponding well to the rising 20E titer in the hemolymph. The level of DNA synthesis in culture was further elevated when 20% hemolymph was added together with 20E, suggesting that other direct stimulatory factors for allatal DNA synthesis may also exist in the hemolymph.

Cyclic juvenile hormone biosynthesis in the cockroach, Blattella germanica: effects of ovariectomy and corpus allatum denervation.

The corpora allata (CA) of ovariectomized adult Blattella germanica females exhibited delayed but high rates of juvenile hormone biosynthesis in vitro. Using the onset of sexual receptivity as a probe of the degree of CA activation in females, we demonstrated at least one cycle of CA activity in the experimentally synchronized ovariectomized females. Following their activation, the CA exhibited a partial and transient decline in activity, but in contrast to the CA of intact females, this decline was not accompanied by a regression in CA volume. CA of intact and ovariectomized females that were denervated from the brain were activated, but the subsequent decline in CA activity at the end of the cycle was prevented in ovariectomized females. The presence of an egg-case suppressed the reactivation of the inactive CA in intact females but not in CA-denervated females. We conclude that activation of the CA in B. germanica is not dependent upon either the presence of the ovary or intact nervous connections between the CA and the brain. The brain exerts a partial inhibition on CA activity through intact nerves which is relieved (by disinhibition) in the presence of a young ovary but is enhanced and sustained in the presence of the egg-case. Inhibition of the CA also occurs independently of nervous connections with the brain through factors that originate in the mature ovary and affect both CA activity and morphology.

Sexual differentiation of nymphal corpora allata and the effects of ovariectomy on adult gland morphometrics in Blattella germanica.

Changes in the number of corpus allatum (CA) cells were investigated in nymphs and in intact and ovariectomized adult female Blattella germanica. The CA of intact adult females exhibit cyclic changes in volume in relation to juvenile hormone (JH) synthesis, while the CA of ovariectomized females become significantly hypertrophied as a result of a gradual and continuous increase in volume that is independent of JH biosynthesis. In both intact and ovariectomized females changes in JH synthesis and CA volume are not related to total cell number which remains relatively constant. However, adult females have twice as many CA cells as do adult males as a result of a female-specific increase in total cell number late in the last nymphal instar.

Mitogenic effects of 20-hydroxyecdysone on neurogenesis in adult mushroom bodies of the cockroach, Diploptera punctata.

The purpose of this study was to examine the mitogenic effects of 20-hydroxyecdysone on neurogenesis in mushroom bodies of the adult cockroach, Diploptera punctata. The occurrence of neurogenesis was studied immunocytochemically after in vivo labeling with 5-bromo-2'-deoxyuridine (BrdU). The number of BrdU-labeled cells in the mushroom bodies was high shortly after adult ecdysis, then gradually decreased, and proliferation ceased on day 8. 20-Hydroxyecdysone injection during the early adult stages significantly delayed the decrease in mitotic activity. Moreover, 20-hydroxyecdysone injection during the late stage stimulated quiescent mushroom body neuroblasts to initiate their mitotic activity in a dose-dependent manner. These results indicated that the mushroom body neuroblasts of this insect become quiescent in the maturing central nervous system, but retain the capacity for proliferation if exposed to appropriate environmental signals. We conclude that 20-hydroxyecdysone has a mitogenic effect on neurogenesis in mushroom bodies of this insect.

Association of protein phosphatase 2A with its substrate vimentin intermediate filaments in 9L rat brain tumor cells.

The importance of protein phosphatases in maintaining the integrity of intermediate filaments is supported by the fact that intermediate filaments would undergo a massive reorganization in cells treated with inhibitors of protein phosphatases 1 and 2A. Herein we used okadaic acid to investigate the differential roles of protein phosphatases 1 and 2A in the maintenance of intermediate filament integrity in 9L rat brain tumor cells. Protein phosphatase 2A activity was substantially inhibited after treatment with 400 nM okadaic acid for 2 h, whereas the activity of protein phosphatase 1 was only slightly affected. Furthermore, protein phosphatase 2A shows selective specificity toward phosphovimentin, which was immunologically precipitated from isotopically labeled and okadaic acid-treated cells. Further biochemical fractionation and microscopic studies revealed that vimentin intermediate filaments were colocalized with protein phosphatase 2A, but not protein phosphatase 1, in control cells. On okadaic acid treatment, vimentin filament disassembled and protein phosphatase 2A redistributed throughout the cytoplasm, suggesting that these two proteins separate from each other, whereas protein phosphatase 2A was inhibited. This working hypothesis was further supported by treatment with a low concentration (40 nM) of okadaic acid, which causes the same phenomenon. Taken together, our results showed that protein phosphatase 2A could be assigned to the intermediate filaments to serve the physiological role in maintaining the proper phosphorylation level of intermediate filaments in normal cells. This finding should pave the way for the elucidation of the regulatory mechanism of intermediate filament organization governed by protein phosphorylation.