Fibrillar Inclusions Research Articles

Molecular crowding accelerates fibrillization of alpha-synuclein: could an increase in the cytoplasmic protein concentration induce Parkinson's disease?

Parkinson's disease (PD) is one of many neurodegenerative diseases that are characterized by amyloid fibril formation. Alpha-synuclein is a primary component of the fibrillar neuronal inclusions, known as Lewy bodies, that are diagnostic of PD. In addition, the alpha-synuclein gene is linked to familial PD. Fibril formation by alpha-synuclein proceeds via discrete beta-sheet-rich oligomers, or protofibrils, that are consumed as fibrils grow. Both FPD mutations accelerate formation of protofibrils, suggesting that these intermediates, rather than the fibril product, trigger neuronal loss. In idiopathic PD, other factors may be responsible for accelerating protofibril formation by wild-type alpha-synuclein. One possible factor could be molecular crowding in the neuronal cytoplasm. We demonstrate here that crowding using inert polymers significantly reduced the lag time for protofibril formation and the conversion of the protofibril to the fibril, but did not affect the morphology of either species. Physiologically realistic changes in the degree of in vitro crowding have significant kinetic consequences. Thus, nonspecific changes in the total cytoplasmic protein concentration, induced by cell volume changes and/or altered protein degradation, could promote formation of and stabilize the alpha-synuclein protofibril.

Similarities between spinocerebellar ataxia type 7 (SCA7) cell models and human brain: proteins recruited in inclusions and activation of caspase-3.

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant polyglutamine disorder presenting with progressive cerebellar ataxia and blindness. The molecular mechanisms underlying the selective neuronal death typical of SCA7 are unknown. We have established SCA7 cell culture models in HEK293 and SH-SY5Y cells, in order to analyse the effects of overexpression of the mutant ataxin-7 protein. The cells readily formed anti-ataxin-7 positive, fibrillar inclusions and small, nuclear electron dense structures. We have compared the inclusions in cells expressing mutant ataxin-7 and in human SCA7 brain tissue. There were consistent signs of ongoing abnormal protein folding, including the recruitment of heat-shock proteins and proteasome subunits. Occasionally, sequestered transcription factors were found. Activated caspase-3 was recruited into the inclusions in both the cell models and human SCA7 brain and its expression was upregulated in cortical neurones, suggesting that it may play a role in the disease process. Finally, on the ultrastructural level, there were signs of autophagy and nuclear indentations, indicative of a major stress response in cells expressing mutant ataxin-7.

Chronic systemic pesticide exposure reproduces features of Parkinson's disease.

The cause of Parkinson's disease (PD) is unknown, but epidemiological studies suggest an association with pesticides and other environmental toxins, and biochemical studies implicate a systemic defect in mitochondrial complex I. We report that chronic, systemic inhibition of complex I by the lipophilic pesticide, rotenone, causes highly selective nigrostriatal dopaminergic degeneration that is associated behaviorally with hypokinesia and rigidity. Nigral neurons in rotenone-treated rats accumulate fibrillar cytoplasmic inclusions that contain ubiquitin and alpha-synuclein. These results indicate that chronic exposure to a common pesticide can reproduce the anatomical, neurochemical, behavioral and neuropathological features of PD.

Detection of a Geminivirus Infecting Sweet Potato in the United States

In 1994, a sweet potato sample showing leaf curl symptoms was collected from the field in Louisiana. When graft-inoculated, Ipomoea nil cv. Scarlett O'Hara reacted with severe leaf distortion and chlorosis symptoms. I. aquatica reacted with a bright yellow mottle. The virus isolated was designated the United States isolate of sweet potato leaf curl virus (SPLCV-US). It was transmitted to I. nil by the sweet potato whitefly, Bemisia tabaci biotype B. DNA probes prepared with component A of pepper Huasteco geminivirus, with an isolate of bean golden mosaic geminivirus from Guatemala, with an isolate of tomato mottle geminivirus from Florida, and with an isolate of tomato yellow leaf curl geminivirus from the Dominican Republic (TYLCV-DR) hybridized with a 2.6-kb DNA band present in DNA extracts from plants infected with SPLCV-US. Probes prepared with the B component of these geminiviruses did not hybridize with these DNA extracts. We were unable to amplify SPLCV-US DNA products by polymerase chain reaction (PCR) in quantities that could be visualized by ethidium bromide staining. However, Southern blots from amplifications with primers AV494/AC1048 revealed PCR products of approximately 600 bp and 550 bp when hybridized with the TYLCV-DR probe. These results were consistently obtained from infected I. cordatotriloba and less consistently from I. aquatica or I. setosa. Fibrillar inclusions were occasionally seen, and granular aggregates of viruslike particles were observed in the nucleus of infected I. cordatotriloba. These results suggest that the virus isolated from sweet potato with leaf curl symptoms belongs to the geminivirus group.

Glial-Neuronal Interactions in Alzheimer Disease

Activated microglia, overexpressing interleukin-1 (IL-1), and activated astrocytes, overexpressing S100beta, have been implicated in the formation and evolution of tau2-immunoreactive (tau2+) neuritic plaques in Alzheimer disease. In this study, we assessed the role of IL-1alpha+ microglia and S100beta+ astrocytes in the pathogenesis of another cardinal histopathological feature of Alzheimer disease: tau2+ neurofibrillary tangles. Four distinct stages of neurofibrillary tangle formation were identified: neurons with granular perikaryal tau2 immunoreactivity (stage 0); fibrillar neuronal inclusions (stage 1); dense, neuronal soma-filling inclusions (stage 2); and acellular, fibrillar deposits (stage 3, "ghost tangles"). The numbers of tangles in randomly selected fields of parahippocampal cortex from 11 Alzheimer patients correlated with both the numbers of IL-1alpha+ microglia and the numbers of S100beta+ astrocytes in these fields (r = 0.72, p < 0.02; r = 0.73, p = 0.01, respectively). There were progressive increases in frequency of association between tangle stages and both IL-1alpha+ microglia and S100beta+ astrocytes: 48, 56, 67, and 92% of stage 0-3 tangles, respectively, had associated IL-1alpha+ microglia; and 21, 37, 55, and 91% of stage 0-3 tangles had associated S100beta+ astrocytes. This progressive association of activated IL-1alpha+ microglia and activated S100beta+ astrocytes with tau2+ tangle stages suggests a role for glial-neuronal interactions in the degeneration of tangle-bearing neurons in Alzheimer disease.

Immunoelectron Microscopic Localization of Peroxisomal Enzymes in the Fibrillar Structures of Rat Liver Peroxisomes Induced by Administration of Acetylsalicylic Acid.

Administration of acetylsalicylic acid (ASA) to rats caused an alteration in intraperoxisomal localization of enzymes in liver peroxisomes as investigated by means of immunoelectron microscopy. Rats were fed on a diet containing 0.5% ASA for 2 weeks and livers were fixed by perfusion. Vibratome sections of the liver tissue were processed to routine electron microscopy (EM), alkaline 3, 3′-diaminobenzidine (DAB) reaction for catalase, and post embedding immunocytochemistry for several peroxisomal enzymes. By light microscopy (LM) of the DAB stained materials, many large spindleform peroxisomes were noted in the cytoplasm of hepatocytes, among which normal size of peroxisomes was also observed. By EM, such large peroxisomes contained fibrillar inclusions in the matrix in addition to the crystalloid core. The fibrillar inclusions were more strongly stained than the matrix after the DAB reaction for catalase. By postembedding immunoelectron microscopy, gold particles showing catalase, acyl-CoA oxidase, L-α-hydroxy-acid oxidase A and serine: pyruvate/glyoxylate aminotransferase (SPT) were closely associated with the fibrillar structures. Urate oxidase was confined exclusively to the crystalloid core, on which a few gold particles showing L-α-hydroxy-acid oxidase A was also noted. D-amino acid oxidase was associated exclusively with a clear spot located adjacent to the crystalloid core. In this clear spot, no other enzymes examined were present. The results show that the fibrillar structure induced by ASA includes at least catalase, acyl-CoA oxidase, L-α-hydroxy-acid oxidase A and SPT, but not urate oxidase and D-amino acid oxidase. D-amino acid oxidase is one of elements that constitute the clear spot. Taken together, the present results demonstrate that some subcompartments exist in the experimentally altered liver peroxisomes.

Association of Chronic Neutrophilic Leukemia and Myeloma with Fibrillar Inclusions in Granulocytes

Association of Chronic Neutrophilic Leukemia and Myeloma with Fibrillar Inclusions in Granulocytes

Assembly of adenovirus-specific nuclear inclusions in lytically infected HeLa cells: an ultrastructural and cytochemical study.

Adenoviruses (Ads) are nuclear DNA viruses that remodel host nuclear structure and function and induce formation of a variety of nuclear inclusions within which Ad DNA is replicated and transcribed. In this study, we have examined inclusion assembly by electron microscopy of samples stained conventionally or with bismuth to detect phosphoproteins. Small dense fibrillar bodies (DFBs) appeared very early associated with interchromatin granule (ICG) clusters. Somewhat later, similar DFBs lay near amorphous, loosely fibrillar structures that were moderately electron dense and showed little bismuth deposition. These clear fibrillar bodies (CFBs) enlarged and DFBs became embedded in their surface. At later stages, CFBs and DFBs were again dissociated. DFBs seen very early were poor in phosphoproteins, but later DFBs, whether embedded in the CFBs or lying near them, were intensely bismuth stained. DFBs and CFBs were less prominent once assembled virions were seen. At this late stage, virions were generally associated with moderately dense, slightly bismuth positive, irregularly shaped fibrillar inclusions that have previously been identified as viral genome storage sites. In addition, very dense fibrillar bodies, consisting usually of an electron-dense fibrillar shell and a less dense fibrogranular core, were observed at all but the earliest stages of infection, often at some distance from CFBs. There was also a major reorganization of host components during infection, including chromatin condensation, reduction of nucleolar volume and aggregation of the fibrillar regions at the nucleolar surface, and increased prominence of ICG clusters. A model is proposed for the assembly of Ad replication factories.

Ultrastructural and cytochemical investigations on the functional role of proteinaceous nuclear inclusions (PNIs) in chlorenchyma plant cells

Summary— Detailed investigations on the fine ultrastructural organization of different forms of proteinaceous nuclear inclusions (PNls) in chlorenchyma plant cells suggest they consist of the same elementary subunits. Previous high magnification of TEM micro‐graphs had shown that the amorphous type of inclusion (A) was mainly composed of elementary fibrils measuring 3.0–3.5 nm in diameter, with no orderly spatial arrangement. New computer image treatments of electron micrographs allowed us to establish that the 8.0–13.0 nm thick filaments — forming the fibrillar (F), crystalline (C) and lamellar (L) inclusions — consist of two elementary fibrils which are coiled in a helix with variable pitch, depending on the type of inclusion. A further secondary coiling of two filaments, about 8.0–9.0 nm in diameter, gives the 20.0–25.0 nm thick tubules which form the characteristic tubular inclusion (T). Correlating the distributive data of PNIs with observations on their ultrastructural morphology and with micrographs of partial aggregation or disgregation patterns of the inclusions, led to the hypothesis that the different forms are not different classes of proteins, but simply different stages of structural complexity of the same protein. To determine whether the intranuclear inclusion protein is nucleolar or nucleolus‐associated, cytochemical and immunocytochemical tests were performed on ultrathin sections or leaf lamina tissue in block. These techniques proved that PNIs do not belong to the class of argyrophilic proteins (AgNOR‐proteins), and particularly not nucleolin and fibrillarin, two of the major nucleolar proteins. Structural similarities to other plant inclusions, especially P‐proteins, and to animal and plant intermediate cytoskeletal filaments (IFs) are discussed with regard to the functional role of PNIs.

Ultrastructural and cytochemical investigations on the functional role of proteinaceous nuclear inclusions (PNIs) in chlorenchyma plant cells

Summary— Detailed investigations on the fine ultrastructural organization of different forms of proteinaceous nuclear inclusions (PNls) in chlorenchyma plant cells suggest they consist of the same elementary subunits. Previous high magnification of TEM micro-graphs had shown that the amorphous type of inclusion (A) was mainly composed of elementary fibrils measuring 3.0–3.5 nm in diameter, with no orderly spatial arrangement. New computer image treatments of electron micrographs allowed us to establish that the 8.0–13.0 nm thick filaments — forming the fibrillar (F), crystalline (C) and lamellar (L) inclusions — consist of two elementary fibrils which are coiled in a helix with variable pitch, depending on the type of inclusion. A further secondary coiling of two filaments, about 8.0–9.0 nm in diameter, gives the 20.0–25.0 nm thick tubules which form the characteristic tubular inclusion (T). Correlating the distributive data of PNIs with observations on their ultrastructural morphology and with micrographs of partial aggregation or disgregation patterns of the inclusions, led to the hypothesis that the different forms are not different classes of proteins, but simply different stages of structural complexity of the same protein. To determine whether the intranuclear inclusion protein is nucleolar or nucleolus-associated, cytochemical and immunocytochemical tests were performed on ultrathin sections or leaf lamina tissue in block. These techniques proved that PNIs do not belong to the class of argyrophilic proteins (AgNOR-proteins), and particularly not nucleolin and fibrillarin, two of the major nucleolar proteins. Structural similarities to other plant inclusions, especially P-proteins, and to animal and plant intermediate cytoskeletal filaments (IFs) are discussed with regard to the functional role of PNIs.

Age-related accumulation of congophilic fibrillar inclusions in endocrine cells

Intracellular fibrillar congophilic inclusions are well known as neurofibrillary tangles in neurons and as Biondi bodies in choroid plexus epithelial cells. Recently similar amyloid-like inclusions in adrenal cortical cells were described (Eriksson and Westermark 1990). This study on 150 adrenal glands confirms these observations. In our material the age-related accumulation of congophilic inclusions starts earlier (in the sixth decade) and reaches a higher incidence (42.7%). We found similar intracellular inclusions in other endocrine organs, for example in the anterior lobe of the pituitary, in the cells of parathyroid glands and in Sertoli cells. The age-related incidence of these fibrillar inclusions in the pituitary was 68%: the co-incidence with interstitial amyloid deposits was 49.5%. Thus the intracellular accumulation of congophilic fibrils in old age is a wide-spread phenomenon and occurs not only in neurons but also in endocrine cells (adrenal, pituitary and parathyroid glands) and in active secretory cells (choroid plexus and Sertoli cells).

The perturbation of the endomembrane network of the chlamydomonad alga Gloeomonas by the herbicide isopropyl N‐phenyl carbamate (IPC)

Abstract. The highly complex and symmetric endomembrane network of the chlamydomonad alga, Gloeomonas kupferri, can be disrupted by the herbicide, isopropyl N‐phenyl carbamate (IPC). Upon incubation with IPC for between 30 min and 1 h, curling of the dictyosomes, proliferation of small vesicles from the trans face and swelling of the peripheral vacuolar complex takes place. Longer treatments result in degeneration of dictyosome cisternae into vesicular masses and the formation of supervacuolar complexes. Enzyme cytochemistry revealed that endomembrane ‘marker’ enzymes are altered/inhibited by IPC. A second but striking I PC‐induced effect is the production of branched, fibrillar inclusions in the cell nucleus. A discussion of I PC‐induced affects on ion regulation and microtubule/MTOC distribution in the cell is presented.

Localization of the adenovirus early region 1B 55-kilodalton protein during lytic infection: association with nuclear viral inclusions requires the early region 4 34-kilodalton protein

The distribution of the adenovirus early region 1B 55-kDa protein (E1B-55kDa) in lytically infected HeLa cells was determined. At the time of infection, when the E1B-55kDa protein facilitates the cytoplasmic accumulation of viral mRNA while simultaneously restricting the accumulation of most cellular mRNA, five distinct intracellular localizations of the protein were observed. Only one of these was disrupted when cells were infected with a mutant virus that fails to produce a second viral protein encoded by early region 4 (E4-34kDa). This protein normally forms a complex with the E1B-55kDa polypeptide, enabling it to influence RNA metabolism. This key localization of the E1B protein was within and about the periphery of nuclear viral inclusion bodies believed to be the site of viral DNA replication and transcription. In the absence of the E4-34kDa protein, the coincidence of E1B-55kDa-specific immunofluorescence and phase-dense viral inclusions was reduced compared with that in a wild-type infection. Similarly, by immunoelectron microscopy, the relative number of E1B-55kDa-specific immunogold particles associated with the clear fibrillar inclusion bodies was reduced. However, the E4-34kDa protein was not required for the close association of the early region 2A DNA binding protein with the viral inclusions. We propose that the viral 55-kDa-34-kDa protein complex interacts with a cellular factor required for cytoplasmic accumulation of mRNAs and directs it to the periphery of the transcriptionally active viral inclusion bodies. This model provides an explanation for the ability of these viral proteins to simultaneously enhance accumulation of viral mRNAs and inhibit accumulation of cellular mRNAs.

Ultrastructural and cytochemical studies of nucleolus-like bodies in neurons of rat sympathetic ganglia

Intracytoplasmic fibrillar inclusions, generally referred to as nucleolus-like bodies (NLBs) were studied by means of ultrastructural cytochemistry. The structure of these bodies was visualized by several different staining procedures: conventional electron microscopy and preferential staining methods for localization of various proteins including ribonucleoproteins, basic proteins, glycoproteins and phosphorylated proteins. The results of the cytochemical tests indicate that NLBs have an essentially proteinaceous nature. They consist of ribonucleoproteins, basic proteins and glycoproteins but do not contain phosphorylated proteins. These findings suggest that NLBs are, at least partially, of the same nature as nucleoli and coiled bodies. The origin of NLBs and their possible functional role is briefly discussed.

ULTRASTRUCTURE OF THE TRANSFER TISSUES DURING VIVIPAROUS SEEDLING DEVELOPMENT IN RHIZOPHORA MANGLE (RHIZOPHORACEAE)

All water and nutrients required for the growth of the huge viviparous seedlings of Rhizophora mangle must be transported from the inner surface of the integument (maternal tissue), across a layer of persistent endosperm cells (seedling tissue), and to the surface of the cylindrical cotyledonary body. We describe the ultrastructure of these tissues at two functionally different stages of embryo and seedling development (i.e., 2–3 wk and 2–3 mo postgermination, respectively). Integumentary cells adjacent to the endosperm have unique plastids and divide more frequently than cells further away from the endosperm/integument interface. Later, fibrillar inclusions develop in vacuoles of the integument cells. The outermost layer of endosperm cells differentiates into transfer cells with wall ingrowths on the external walls, and the remainder of the endosperm degenerates during the embryo to seedling transition. The wall ingrowths gradually occlude the lumena of the transfer cells, a process which is complete long before the seedling abscises; therefore, the seedling may be photosynthetically independent throughout most of its development. The outer surface of the seedling cotyledonary body is minimally papillate and exhibits several ultrastructural specializations: centripetal degeneration of external walls which are bounded by electron‐dense deposits; numerous mitochondria; and plastids of unusual structure with many plastoglobuli, and which contain unique yellow pigments having absorption spectra characteristic of carotenoids. HPLC residence times of these pigments are unlike those of β‐carotene or other common carotenoids. Transfer tissues of Rhizophora may have functions not found in other plants, such as salt exclusion and facilitating viviparous germination, but the ultrastuctural correlates of these functions remain uncertain.

Proteinaceous inclusions in specialized thallus cells of Chylocladia verticillata (Rhodymeniales, Rhodophyta)

Abstract The marine red alga Chylocladia verticillata (Rhodymeniales, Rhodophyta) possesses numerous scattered small cells on the inner face of the hollow thallus which are characterized by the presence ofa great number of cytoplasmic membrane-bound fibrillar inclusions. Application of the Coomassie Brilliant Blue R histochemical technique and treatment of tissue with enzyme solutions indicated that these inclusions are constituted of protein. The frequent, close association of the proteinaceous inclusions with endoplasmic reticulum elements and the occasional coalescence of the endoplasmic reticulum membranes to the inclusion membrane suggest that these inclusions may originate from the endoplasmic reticulum. Fibrillar elements of cytoplasmic inclusions appear to be organized into successive arrays of arcs arranged in nearly parallel stacked levels, as interpreted by examining different planes of inclusion sections. Analysis of the diffraction pattern of such sections has revealed that the proteinaceous ...

Electron microscopic and physicochemical analysis of a reo-like virus of the planthopper Peregrinus maidis.

A virus, determined to be a member of the Reoviridae, was found naturally infecting colonies of Peregrinus maidis, the maize planthopper. The virus, referred to as peregrinus maidis-virus (PgMV), was found by electron microscopy throughout all P. maidis tissues examined, including salivary glands, testes, ovaries, and intestines. The virus particles were associated with fibrillar viroplasmic inclusions in the cytoplasm, of a type characteristic of the Reoviridae. The particles, purified from P. maidis, had morphologies typical of the Reoviridae, with a mean diameter of 68.7 nm. The purified virus contained 12 double-stranded (ds)RNAs, while dsRNA extracts of PgMV-infected P. maidis showed these, plus one additional high-molecular weight dsRNA. No similar dsRNAs were present in extracts of another planthopper. Perkinsiella saccharicida, the corn leafhopper (Dalbulus maidis), or from Zea mays upon which the P. maidis had been reared. Six major and four minor proteins were identified in purified PgMV preparations, but we were unable to determine which of these were definitely of viral or host origin. PgMV was found to be serologically unrelated to three other members of the Reoviridae. The similarities of PgMV to other members of the Reoviridae are discussed.

Ultrastructural characterization of the central component of the medial preoptic nucleus

The fine structure of the sexually dimorphic central part of the medial preoptic nucleus was studied in male and female rats. Neurons of this subnucleus are characterized by having convoluted nuclear membranes and fibrillar nuclear inclusions. Within the perinuclear domain there is a well developed neurosecretory apparatus, i.e., Golgi complexes, assorted types of secretory granules, and lysosome-like bodies, whereas the marginal cytosol contains stacked cisterns of rough endoplasmic reticulum, nucleolus-like inclusions, and few mitochondria. The neuropil is made up of thick dendrites, fascicles of thin unmyelinated axons, and few small myelinated axons. Additionally four types of synaptic boutons were found according to the morphology of their vesicular contents. No apparent qualitative sex differences were noted. These cytologic features which suggest secretory activity, coupled with reports that have shown the presence of several neurohormones and peptides within the central part of the medial preoptic nucleus lend further support to the neuroendocrine nature of these neurons.

Ultrastructural Study of Oogenesis and Fertilization inSycon ciliatum(Porifera, Calcispongiae)

Summary The oogenesis of the calcareoous sponge Sycon ciliatum has been studied by electron microscopy. In this species, oogonia probably derive from choanocytes through loss of collar and flagellum and formation of phagosome-like inclusions. Oogonia can be occasionally found within flagellated chambers and show a prominent rough endoplasmic reticulum, several mitochondria and polygonal dense granules. The latter are also visible in choanocytes. Oocytes lie in the mesohyl beneath the choanoderm. They contain a nucleolated nucleus, vesiculated granules and phagosome-like inclusions involved in the formation of fibrillar yolk material. Mature eggs are large, irregularly shaped, and filled with fibrillar yolk inclusions. A second ultrastructural confirmation of the carrier-cell mediated fertilization of calcareous sponges is also given.

Immunocytochemical investigation of the causal agent of cassava mosaic disease in southern Africa

Cassava mosaic disease (CMD) exists throughout Africa, and cassava latent virus (CLV) has been implicated as the etiological agent in Kenya and West Africa. However, in Southern Africa, the causal agent of CMD was not until recently associated with CLV, and the possibility of a second flexuous virus particle has not been ignored. Attempts to isolate and visualize CLV antigen have been successful with Nicotiana benthamiana, an indicator host plant of CLV, but all efforts to isolate and visualize particles in infected cassava plants have failed. Immunocytochemical studies were undertaken in an attempt to localize virus antigen in infected cassava tissue. Cytochemical staining (light microscope) of infected cassava leaf material revealed the presence of inclusion bodies in epidermal and palaside mesophyll cells, and in epidermal collenchyma and outer parenchyma cells from the petiole and stem. However, transmission electron-microscopical (TEM) investigations revealed electron dense bodies in the cytoplasm, and no characteristic CLV nuclear inclusion bodies were evident. Transmission experiments to N. benthamiana and N. tabacum were attempted and leaves, exhibiting symptoms, examined microscopically. The nuclei appeared swollen (in comparison to uninfected leaves), a characteristic of CLV- infected N. benthamiana. However at the TEM level, no characteristic fibrillar-ring inclusion bodies or particles, could be visualized. Further immunocytochemical investigations were initiated, employing antisera raised against CLV isolated from N. benthamiana, and antisera for cassava common mosaic virus (CCMV), cassava brown streak virus (CBSV) and cassava X virus (CsXV). Goat anti-rabbit IgG-gold was used as a direct stain. No labelling occurred with CCMV and CBSV antisera. Intense gold labelling was located in the cytoplasm of phloem, mesophyll and epidermal cells of infected cassava and to a lesser extent in N. tabacum and N. benthamiana using affinity chromatography purified CLV antiserum. Little labelling was observed in nuclei of infected cells. Inconclusive results were obtained with CsXV antiserum. Immunogold labelling located CLV viral antigens in infected cassava leaf tissue. This observation, together with positive ELISA, transmission and DNA hybridization experiments, proves conclusively that CLV viral antigen is present in infected cassava in Southern Africa. However, most viral antigen in infected cassava, unlike N. benthamiana (fibrillar and granular nuclear inclusions) appears to be in the cytoplasm. This may tentatively suggest that the CLV protein is synthesized in the cytoplasm of its natural host, cassava, even though the virus may assemble in the nucleus at the appropriate time. However, as yet no virus inclusions have been observed in nuclei of infected cassava. Due to previous isolation of a flexuous rod and ambiguous staining results, the possibility of two viruses in cassava cannot be ruled out.