In search of a function for centrins.

Spindle pole bodies, basal bodies and centrosomes are morphologically quite different structures that nevertheless perform similar microtubule-organizing functions in diverse cell types. The recent discoveries that both centrins and gamma-tubulin are common components of these structures suggest a molecular basis for their common functions. The role of centrins is just beginning to be investigated. These filament-associated proteins bind Ca2+. The filaments contract at least in certain circumstances by an ATP-independent mechanism. However, yeast centrin is clearly involved in the duplication of the spindle pole body. A common molecular mechanism may underlie these two apparently different functions.

Basal-body-associated macromolecules: a continuing debate.

Controversy over the possibility that centrioles/basal bodies contain nucleic acids has overshadowed results demonstrating other macromolecules in the lumen of these organelles. Glycogen particles, which are known to be present within the lumen of the centriole/basal body of sperm cells, have now been found in basal bodies of protists belonging to three different groups. Here, we extend the debate on a role for RNA in basal body/centriole function and speculate on the origin and the function of centriolar glycogen.

Isolation of nuclear envelopes with polyanions.

Optimal conditions for the isolation of nuclear envelopes by the action of heparin on nuclei are established and a morphological and biochemical study of such isolated envelopes is presented. An almost 100% yield of pure nuclear envelopes can be obtained by a single sedimentation step after incubation of nuclei with heparin for 40 min at 4 degrees C. The nuclear membrane pellet obtained in this way contains whole envelopes with a preserved perinuclear space and with ribosomes present on the outher leaflet. A single band with an apparent buoyant density of 1.18 is obtained by sucrose density gradient analysis. The chemical composition of the pellet is similar to that of the purified membranes and corresponds to 62% proteins, 34% phospholipids, 3% RNA, and 0.5% DNA. The presence of low concentrations of sodium phosphate (2-10 mM) is critical for a complete solubilization of the chromatin. A less rapid and complete solubilization is obtained with the potassium salt. Low concentrations of Mg++ (1-3 mM) counteract chromatin solubilization by heparin mainly at the level of chromatin-nuclear membrane association. The presence of EDTA in the medium leads to isolated nuclear envelopes on which neither ribosomes nor nuclear pores are visible, indicating the pore structure is dependent on the presence of Ca++ or Mg++. A comparison with other polyanions indicates a decisive advantage of heparin. However, pure nuclear envelopes can also be obtained by the action of dextran sulfate (mol wt 500,000) on nuclei incubated for 5 min at 37 degrees C, in the presence of phosphate ions.

The cortical microfilament system of lymphoblasts displays a periodic oscillatory activity in the absence of microtubules: implications for cell polarity.

For an understanding of the role of microtubules in the definition of cell polarity, we have studied the cell surface motility of human lymphoblasts (KE37 cell line) using video microscopy, time-lapse photography, and immunofluorescent localization of F-actin and myosin. Polarized cell surface motility occurs in association with a constriction ring which forms on the centrosome side of the cell: the cytoplasm flows from the ring zone towards membrane veils which keep protruding in the same general direction. This association is ensured by microtubules: in their absence the ring is conspicuous and moves periodically back and forth across the cell, while a protrusion of membrane occurs alternately at each end of the cell when the ring is at the other. This oscillatory activity is correlated with a striking redistribution of myosin towards a cortical localization and appears to be due to the alternate flow of cortical myosin associated with the ring and to the periodic assembly of actin coupled with membrane protrusion. The ring cycle involves the progressive recruitment of myosin from a polar accumulation, or cap, its transportation across the cell and its accumulation in a new cap at the other end of the cell, suggesting an assembly-disassembly process. Inhibition of actin assembly induces, on the other hand, a dramatic microtubule-dependent cell elongation with definite polarity, likely to involve the interaction of microtubules with the cell cortex. We conclude that the polarized cell surface motility in KE37 cells is based on the periodic oscillatory activity of the actin system: a myosin-powered equatorial contraction and an actin-based membrane protrusion are concerted at the cell level and occur at opposite ends of the cell in absence of microtubules. This defines a polarity which reverses periodically as the ring moves across the cell. Microtubules impose a stable cell polarity by suppressing the ring movement. A permanent association of the myosin-powered contraction and the membrane protrusion is established which results in the unidirectional activity of the actin system. Microtubules exert their effect by controlling the recruitment of cytoplasmic myosin into the cortex, probably through their direct interaction with the cortical microfilament system.

A human centrosomal protein is immunologically related to basal body-associated proteins from lower eucaryotes and is involved in the nucleation of microtubules.

Isolation of centrosomes from human cells has revealed a proteic pattern which is both complex and specific. As the most prominent structural element of centrosomes in animal cells, the centriole which is present as two copies, is a highly conserved structure, we have attempted to identify centrosomal proteins on the basis of immunocross-reaction with proteins identified in basal bodies from lower eucaryotes. We report that two antibodies, one raised against the Ca(+)-binding protein centrin (Salisbury, J. L., A. T. Baron, B. Surek, and M. Melkonian. 1984. J. Cell Biol. 99:962-970) and the other directed against a 230-kD protein isolated from the infraciliary cytoskeletal lattice of the protozoan Polyplastron m., decorate the centrosome of human cultured cells, and identify one of the major centrosomal components revealed as a doublet of 62/64 kD. Moreover the nucleation reaction of microtubules, which can be efficiently produced on isolated centrosomes, is blocked by the antibodies, a result which strongly implicates the 62/64-kD protein in this centrosomal activity. We also show that the 62/64-kD protein remains insoluble in conditions (0.5 M KI or 8 M urea) which are capable of extracting most of the centrosomal proteins. Immunocytochemical localization by EM of isolated centrosomes revealed the association of this 62/64-kD doublet with the intercentriolar link and the pericentriolar lattice. Our results suggest that conservation of structure in the centrosome from divergent organisms could be matched by conservation of proteins and activity, evidence for the maintenance of a specific function, which could involve Ca2+, associated with the microtubule organizing centers.

Fate of microtubule-organizing centers during myogenesis in vitro.

Microtubule organization and nucleation were studied during in vitro human myogenesis by immunocytology that used monoclonal and polyclonal antitubulin antibodies and a rabbit nonimmune serum that reacts with human centrosomes. In myoblasts, we observed a classical microtubule network centered on juxtanuclear centrosomes. Myotubes possessed numerous microtubules organized in parallel without any apparent nucleation centers. Centrosomes in these cells were not associated one to each nucleus but were often clustered in the vicinity of nuclei groups. They were significantly smaller than those of the mononucleated cells. The periphery of each nucleus in myotubes was labeled with the serum that labels centrosomes suggesting a profound reorganization of microtubule-nucleating material. Regrowth experiments after Nocodazole treatment established that microtubules were growing from the periphery of the nuclei. The redistribution of nucleating material was shown to take place early after myoblast fusion. Such a phenomenon appears to be specific to myogenic differentiation in that artificially induced polykaryons behaved differently: the centrosomes aggregated to form only one or a few giant nucleating centers and the nuclei did not participate directly in the nucleation of microtubules. The significance of these results is discussed in relation to the possible role of the centrosome in establishing cell polarity.

The intercentriolar linkage is critical for the ability of heterologous centrosomes to induce parthenogenesis in Xenopus.

Centrosomes isolated from various sources, including human cells, have the capacity to induce parthenogenetic development when injected into unfertilized amphibian eggs. We recently isolated calf thymus centrosomes and showed that they differ structurally and functionally from previously isolated centrosomes of KE37 cells, in that the two centrioles in calf thymocytes are linearly associated by their proximal ends through a mass of electron dense material and nucleate few microtubules from their distal ends (Komesli, S., F. Tournier, M. Paintrand, R. Margolis, D. Job, and M. Bornens. 1989. J. Cell Biol. 109:2869-2878). We report here that these centrosomes are also unable to induce egg cleavage and examine the various possibilities which could account for this lack of competence. The results show that: (a) the kinetics of microtubule assembly on calf thymus centrosomes in Xenopus extracts are comparable to those of KE37 centrosomes; (b) centrosomes isolated from thymus of calves raised under controlled conditions (without anabolic agents) also lack competence; (c) centrosomes isolated from bovine cells of other tissues are competent; (d) centrosomes isolated from thymus of three other species (rat, mouse, and human) are competent. Since the lack of activity of calf thymus centrosomes apparently was not linked to species or tissue differences, we compared the ultrastructure of the centrosomes in the various centrosome preparations. The results show a strict correlation between the linear arrangement of centrioles and the lack of activity of the centrosomes. They suggest that the centrosome cycle can be blocked when the centrioles are prevented from separating into a nonlinear configuration, a step which might be critical for the initiation of procentriole budding. They also indicate that the centrosome may be involved in the G0-G1 transition.

The Golgi apparatus remains associated with microtubule organizing centers during myogenesis.

In vitro myogenesis involves a dramatic reorganization of the microtubular network, characterized principally by the relocalization of microtubule nucleating sites at the surface of the nuclei in myotubes, in marked contrast with the classical pericentriolar localization observed in myoblasts (Tassin, A. M., B. Maro, and M. Bornens, 1985, J. Cell Biol., 100:35-46). Since a spatial relationship between the Golgi apparatus and the centrosome is observed in most animal cells, we have decided to follow the fate of the Golgi apparatus during myogenesis by an immunocytochemical approach, using wheat germ agglutinin and an affinity-purified anti-galactosyltransferase. We show that Golgi apparatus in myotubes displays a perinuclear distribution which is strikingly different from the polarized juxtanuclear organization observed in myoblasts. As a result, the Golgi apparatus in myotubes is situated close to the microtubule organizing center (MTOC), the cis-side being situated at a fixed distance from the nuclear envelope, a situation which suggests the existence of a structural association between the Golgi apparatus and the nuclear periphery. This is supported by experiments of microtubule depolymerization by nocodazole, in which a minimal effect was observed on Golgi apparatus localization in myotubes in contrast with the dramatic scattering observed in myoblasts. In both cell types, electron microscopy reveals that microtubule disruption generates individual dictyosomes; this suggests that the connecting structures between dictyosomes are principally affected. This structural dependency of the Golgi apparatus upon microtubules is not apparently accompanied by a reverse dependency of MTOC structure or function upon Golgi apparatus activity. Golgi apparatus modification by monensin, as effective in myotubes as in myoblasts, is without apparent effect on MTOC localization or activity and on microtubule stability. The main result of our study is to show that in a cell type where the MTOC is dissociated from centrioles and where antero-posterior polarity has disappeared, the association between the Golgi apparatus and the MTOC is maintained. The significance of such a tight association is discussed.

Characterization of the human homologue of the yeast spc98p and its association with gamma-tubulin.

A trimeric complex formed by Tub4p, the budding yeast gamma-tubulin, and the two spindle pole body components, Spc98p and Spc97p, has recently been characterized in Saccharomyces cerevisiae. We reasoned that crucial functions, such as the control of microtubule nucleation, could be maintained among divergent species. SPC98-related sequences were searched in dbEST using the BLASTN program. Primers derived from the human expressed sequence tag matching SPC98 were used to clone the 5' and 3' cDNA ends by rapid amplification of cDNA ends (RACE)-PCR. The human Spc98 cDNA presents an alternative splicing at the 3' end. The deduced protein possesses 22% identity and 45% similarity with the yeast homologue. We further report that the human Spc98p, like gamma-tubulin, is concentrated at the centrosome, although a large fraction is found in cytosolic complexes. Sucrose gradient sedimentation of the cytosolic fraction and immunoprecipitation experiments demonstrate that both gamma-tubulin and HsSpc98p are in the same complex. Interestingly, Xenopus sperm centrosomes, which are incompetent for microtubule nucleation before their activation in the egg cytoplasm, were found to contain similar amounts of both Spc98p and gamma-tubulin to human somatic centrosomes, which are competent for microtubule nucleation. Finally, affinity-purified antibodies against Spc98p inhibit microtubule nucleation on isolated centrosomes, as well as in microinjected cells, suggesting that this novel protein is indeed required for the nucleation reaction.

Mass isolation of calf thymus centrosomes: identification of a specific configuration.

Centrosomes from calf thymocytes were isolated using a simple preparative procedure that provides large yields of free organelles. A comparative study with centrosomes isolated from human cultured lymphoblasts has led to the discovery of important differences in the structure of the two isolates and in their capacity to nucleate microtubules from purified tubulin. The possibility that the centrosomal structure depends upon the growth state of cells is discussed.

Myosin at the apical pole of ciliated epithelial cells as revealed by a monoclonal antibody.

A monoclonal antibody (CC-212), obtained in a fusion experiment in which basal bodies from quail oviduct were used as immunogen, has been shown to label the apical pole of ciliated cells and to react with a 200-kD protein. This monoclonal antibody was demonstrated to be an anti-myosin from smooth muscle or from nonmuscular cells using the following criteria: On Western blots it reacted with the myosin heavy chains from gizzard and platelet extracts and from cultured cell line extracts, but did not react with striated muscle myosin heavy chains. By immunofluorescence it decorated the stress fibers of well-spread cells with a characteristic striated pattern, while it did not react with myotubes containing organized myofibrils. On native ciliated cells as well as on Triton-extracted ciliated cortices from quail oviduct, this monoclonal antibody decorated the apical pole with a stronger labeling of the periphery of the apical area. Ultrastructural localization was attempted using the immunogold technique on the same preparation. Myosin was associated with a filamentous material present between striated rootlets and the proximal extremities of the basal bodies. No labeling of the basal body itself or of axoneme was observed.

Parthenogenesis in Xenopus eggs requires centrosomal integrity.

Xenopus eggs are laid arrested at second metaphase of meiosis lacking a functional centrosome. Upon fertilization, the sperm provides the active centrosome that is required for cleavage to occur. The injection of purified centrosomes mimics fertilization and leads to tadpole formation (parthenogenesis). In this work we show that the parthenogenetic activity of centrosomes is inactivated by urea concentrations higher than 2 M. The loss of activity is correlated with a progressive destruction of the centriolar cylinder and extraction of proteins. This shows that centrosomes are relatively sensitive to urea since complete protein unfolding and solubilization of proteins normally occurs at urea concentrations as high as 8-10 M. When present, the parthenogenetic activity is always associated with a pelletable fraction showing that it cannot be solubilized by urea. The parthenogenetic activity is progressively inactivated by salt concentrations higher than 2 M (NaCl or KCl). However, only a few proteins are extracted by these treatments and the centrosome ultrastructure is not affected. This shows that both parthenogenetic activity and centrosomal structure are resistant to relatively high ionic strength. Indeed, most protein structures held by electrostatic forces are dissociated by 2 M salt. The loss of parthenogenetic activity produced at higher salt concentrations, while the structure of the centrosome is unaffected, is an apparent paradox. We interpret this result as meaning that the native state of centrosomes is held together by forces that favor functional denaturation by high ionic strength. The respective effects of urea and salts on centrosomal structure and activity suggest that the centrosome is mainly held together by hydrogen and hydrophobic bonds. The in vitro microtubule nucleating activity of centrosomes can be inactivated at salt or urea concentrations that do not affect the parthenogenetic activity. Since egg cleavage requires the formation of microtubule asters, we conclude that the extracted or denatured microtubule nucleating activity of centrosomes can be complemented by components present in the egg cytoplasm. Both parthenogenetic and microtubule nucleating activities are abolished by protease treatments but resist nuclease action. Since we find no RNA in centrosomes treated by RNase, they probably do not contain a protected RNA. Taken together, these results are consistent with the idea that the whole or part of the centrosome structure acts as a seed to start the centrosome duplication cycle in Xenopus eggs.

GMAP-210, A cis-Golgi network-associated protein, is a minus end microtubule-binding protein.

We report that a peripheral Golgi protein with a molecular mass of 210 kD localized at the cis-Golgi network (Rios, R.M., A.M. Tassin, C. Celati, C. Antony, M.C. Boissier, J.C. Homberg, and M. Bornens. 1994. J. Cell Biol. 125:997-1013) is a microtubule-binding protein that associates in situ with a subpopulation of stable microtubules. Interaction of this protein, now called GMAP-210, for Golgi microtubule-associated protein 210, with microtubules in vitro is direct, tight and nucleotide-independent. Biochemical analysis further suggests that GMAP-210 specifically binds to microtubule ends. The full-length cDNA encoding GMAP-210 predicts a protein of 1, 979 amino acids with a very long central coiled-coil domain. Deletion analyses in vitro show that the COOH terminus of GMAP-210 binds to microtubules whereas the NH2 terminus binds to Golgi membranes. Overexpression of GMAP-210-encoding cDNA induced a dramatic enlargement of the Golgi apparatus and perturbations in the microtubule network. These effects did not occur when a mutant lacking the COOH-terminal domain was expressed. When transfected in fusion with the green fluorescent protein, the NH2-terminal domain associated with the cis-Golgi network whereas the COOH-terminal microtubule-binding domain localized at the centrosome. Altogether these data support the view that GMAP-210 serves to link the cis-Golgi network to the minus ends of centrosome-nucleated microtubules. In addition, this interaction appears essential for ensuring the proper morphology and size of the Golgi apparatus.

Centriole disassembly in vivo and its effect on centrosome structure and function in vertebrate cells.

Glutamylation is the major posttranslational modification of neuronal and axonemal tubulin and is restricted predominantly to centrioles in nonneuronal cells (Bobinnec, Y., M. Moudjou, J.P. Fouquet, E. Desbruyères, B. Eddé, and M. Bornens. 1998. Cell Motil. Cytoskel. 39:223-232). To investigate a possible relationship between the exceptional stability of centriole microtubules and the compartmentalization of glutamylated isoforms, we loaded HeLa cells with the monoclonal antibody GT335, which specifically reacts with polyglutamylated tubulin. The total disappearance of the centriole pair was observed after 12 h, as judged both by immunofluorescence labeling with specific antibodies and electron microscopic observation of cells after complete thick serial sectioning. Strikingly, we also observed a scattering of the pericentriolar material (PCM) within the cytoplasm and a parallel disappearance of the centrosome as a defined organelle. However, centriole disappearance was transient, as centrioles and discrete centrosomes ultimately reappeared in the cell population. During the acentriolar period, a large proportion of monopolar half-spindles or of bipolar spindles with abnormal distribution of PCM and NuMA were observed. However, as judged by a quasinormal increase in cell number, these cells likely were not blocked in mitosis. Our results suggest that a posttranslational modification of tubulin is critical for long-term stability of centriolar microtubules. They further demonstrate that in animal cells, centrioles are instrumental in organizing centrosomal components into a structurally stable organelle.

The respective contributions of the mother and daughter centrioles to centrosome activity and behavior in vertebrate cells.

We have generated several stable cell lines expressing GFP-labeled centrin. This fusion protein becomes concentrated in the lumen of both centrioles, making them clearly visible in the living cell. Time-lapse fluorescence microscopy reveals that the centriole pair inherited after mitosis splits during or just after telophase. At this time the mother centriole remains near the cell center while the daughter migrates extensively throughout the cytoplasm. This differential behavior is not related to the presence of a nucleus because it is also observed in enucleated cells. The characteristic motions of the daughter centriole persist in the absence of microtubules (Mts). or actin, but are arrested when both Mts and actin filaments are disrupted. As the centrioles replicate at the G1/S transition the movements exhibited by the original daughter become progressively attenuated, and by the onset of mitosis its behavior is indistinguishable from that of the mother centriole. While both centrioles possess associated gamma-tubulin, and nucleate similar number of Mts in Mt repolymerization experiments. during G1 and S only the mother centriole is located at the focus of the Mt array. A model, based on differences in Mt anchoring and release by the mother and daughter centrioles, is proposed to explain these results.

A peripheral protein associated with the cis-Golgi network redistributes in the intermediate compartment upon brefeldin A treatment.

Human autoantibodies offer unique tools for the study of cellular constituents since they usually recognize highly conserved components, the most difficult to detect due to their low immunogenicity. The serum from a patient with Sjögren's syndrome (RM serum) showing a very high reactivity to the Golgi complex has been shown to immunoprecipitate and to immunodetect by Western blotting experiments a protein mol wt 210,000 (p210) that was shown to be peripheral and cytoplasmically disposed. A close examination of the p210 labeling revealed some differences with Golgi markers: RM serum staining was slightly more extensive than several Golgi markers and showed a discontinuous or granular appearance. Nocodazole induced a specific and early segregation of many p210-associated vesicles or tubules from Golgi apparatus. Upon brefeldin A treatment, p210 did not redistribute in the ER as did other Golgi proteins. In contrast, it exhibited a vesicular pattern reminiscent to that displayed by proteins residing in the intermediate compartment. Double staining immunofluorescence using the RM serum and the marker of the intermediate compartment, p58, revealed segregation of both proteins in control conditions but colocalization in BFA-treated cells. We have further demonstrated by combining different drug treatments that p210-containing elements in brefeldin A-treated cells belong indeed to the intermediate compartment. Experiments on brefeldin A recovery suggested that these p210 elements might play a role in reformation and repositioning of the Golgi apparatus. Ultrastructural localization performed by immunoperoxidase staining allowed us to establish that p210 interacted with the external side of an abundant tubulo-vesicular system on the cis side of the Golgi complex which extended to connecting structures and vesicles between saccules or stacks of cisternae, p210 appears to be a novel protein residing in the cis-Golgi network that may cycle between the Golgi apparatus and the intermediate compartment.

A role for centrin 3 in centrosome reproduction.

Centrosome reproduction by duplication is essential for the bipolarity of cell division, but the molecular basis of this process is still unknown. Mutations in Saccharomyces cerevisiae CDC31 gene prevent the duplication of the spindle pole body (SPB). The product of this gene belongs to the calmodulin super-family and is concentrated at the half bridge of the SPB. We present a functional analysis of HsCEN3, a human centrin gene closely related to the CDC31 gene. Transient overexpression of wild-type or mutant forms of HsCen3p in human cells demonstrates that centriole localization depends on a functional fourth EF-hand, but does not produce mitotic phenotype. However, injection of recombinant HsCen3p or of RNA encoding HsCen3p in one blastomere of two-cell stage Xenopus laevis embryos resulted in undercleavage and inhibition of centrosome duplication. Furthermore, HsCEN3 does not complement mutations or deletion of CDC31 in S. cerevisiae, but specifically blocks SPB duplication, indicating that the human protein acts as a dominant negative mutant of CDC31. Several lines of evidence indicate that HsCen3p acts by titrating Cdc31p-binding protein(s). Our results demonstrate that, in spite of the large differences in centrosome structure among widely divergent species, the centrosome pathway of reproduction is conserved.

Centrosome-dependent exit of cytokinesis in animal cells.

As an organelle coupling nuclear and cytoplasmic divisions, the centrosome is essential to mitotic fidelity, and its inheritance could be critical to understanding cell transformation. Investigating the behavior of the centrosome in living mitotic cells, we documented a transient and remarkable postanaphase repositioning of this organelle, which apparently controls the release of central microtubules from the midbody and the completion of cell division. We also observed that the absence of the centrosome leads to defects in cytokinesis. Together with recent results in yeasts, our data point to a conserved centrosome-dependent pathway that integrates spatial controls into the decision of completing cell division, which requires the repositioning of the centrosome organelle.

Tissue-specific expression and subcellular localisation of mammalian delta-tubulin.

The properties of the microtubule network are regulated at various levels including tissue-dependent isotype switching, post-translational modification of alpha- and beta-tubulin, and by a variety of microtubule-associated molecules (for reviews, see [1-3]). Microtubule nucleation is attributed to gamma-tubulin, which is present in protein complexes at the centrosome and in the cytoplasm [4,5]. A screen for flagellar mutants in the green alga Chlamydomonas reinhardtii has led to the identification of a fourth member of the tubulin gene superfamily, delta-tubulin. In this unicellular organism, the lack of a functional delta-tubulin gene copy causes aberrant numbers of flagella, depending on the age of the corresponding basal bodies; mutants also show abnormal ultrastructure of the basal bodies and a misplacement of the cleavage furrow at mitosis [6]. Here, we report the isolation of the mouse delta-tubulin homologue and show that the gene is highly expressed in testis. In the elongating spermatid, delta-tubulin associated with the manchette, a specialised microtubule system present during reshaping of the sperm head. The protein specifically localised at the perinuclear ring of the manchette, at the centriolar vaults and along the principal piece of the sperm flagellum. In somatic cell lines, unlike most other tubulins, mammalian delta-tubulin was both cytoplasmic and nuclear and did not colocalise with microtubules. The protein was enriched at the spindle poles during mitosis and we found that gamma-tubulin coimmunoprecipitated with delta-tubulin. Together, the data indicate a specialised role for mammalian delta-tubulin that is distinct from other known tubulins.

Increase in the expression of a family of small guanosine triphosphate-binding proteins, rab proteins, during induced phagocyte differentiation.

Rab is a newly identified family of small G-proteins that share 35-70% homology with the yeast Sec4p and Ypt1p involved in the regulation of the secretory pathway. Mature phagocytes display functions requiring organized intracellular traffic and, for this reason, we questioned whether phagocyte differentiation could correlate with the increased expression of rab proteins. Rabbit antisera raised against the recombinant proteins rab1Ap, 2p, 4p, and 6p were able to detect the corresponding proteins in the human monoblast leukemic cell line U937. When these cells were induced to differentiate into monocyte/macrophage-like cells displaying functional characteristics of a normal phagocyte, rab1Ap, 2p, 4p, and 6p were increased and this correlated with an increase in the rab transcripts. Using a rab5 probe, we also observed an increased expression of the rab5 gene in differentiated cells. Similarly, differentiation of the human leukemic myeloblast HL60 cell line along either monocyte or granulocyte pathways induced an increased expression of the rab proteins. Rab proteins were also detected in human neutrophils and in guinea pig alveolar macrophages. As degranulation is one of the phagocyte functions acquired in the late stage of differentiation, we investigated whether rab proteins would be involved in this process. Although rab proteins were tightly membrane bound, none of them was detected in the specific or azurophil granules purified from human neutrophils. The increased expression of rab proteins in mature phagocytes suggests that they may promote functions highly developed in these cells.