Centrin is a component of the periocentriola lattice

Biol Cell (1992) 76, 383-388

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© Elsevier, Paris Original

article

Centrin is a component of the pericentriolar lattice Andre

T B a r o n a, T a m m y

M Greenwood

a, C h r i s t o p h e r

W B a z i n e t b, J e f f r e y L S a l i s b u r y

a

aLaboratory for Cell Biology, Department of Biochemistry and Molecular Biology, Mayo Clinic Foundation, Rochester, Minnesota 55905; °Case Western Reserve University, Department of Genetics, Cleveland, Ohio 44106, USA (Received 17 September 1992; accepted 8 January 1993)

Summary - Here, we use three polyclonal anticentrin antisera designated 08/28, 26/14-1, and 26/14-2 to further characterize the

pericentriolar lattice of metazoan cells. All of these antibodies give an indistinguishable localization pattern that consists of a constellation of pericentrosomal spots. In QT6 cells these spots are few in number and closely associated with the centriolar region, whereas in P t K 2 cells they are more numerous and distributed further from the point of microtubule focus. In mitotic cells, centrin is localized to the spindle poles and spindle apparatus. We demonstrate here that the pericentriolar lattice of PtK 2 and QT6 cells is, in part, composed of proteins characterized by acidic pls (4.4 to 5.4), low molecular mass (Mr 18500-21 000), and calcium-binding; these attributes and the immunoreactivity of these proteins to anticentrin antibodies indicate that they are centrin isoforms of metazoan cells. Finally, we confirm our earlier observation that PtK 2 cells contain a centrin-related protein of M r 165000; QT6 cells also contain centrin-related proteins (Mr 64000-165 000). We conclude that centrin is a component of the pericentriolar lattice of higher eukaryotic centrosomes. centrin / centrosome / cytoskeleton I pericentriolar lattice / pericentriolar material

Introduction

Centrin-based filament systems of flagellate algal unicells share a number of characteristics. They are, in part, composed of an acidic low molecular mass (M r = 2 0 0 0 0 21000) calcium-binding protein called centrin, which has phosphorylated isoforms [19, 36]. Molecular cloning of centrin from the alga Chlamydomonas reinhardtii has revealed that centrin belongs to the EF-hand superfamily of calcium-binding proteins, which include calmodulin and parvalbumin [15]. Indirect immunofluorescence microscopy shows that centrin is localized ubiquitously to the algal flagellar basal body apparatus [24, 31-34]. Immunogold cytochemistry and transmission electron microscopy of conventional thin sections reveal that centrin is a component of a specific class of 3 - 8 - n m diameter filaments. These centrin-based filaments belong to a newlyrecognized group of fibers, which are called 'nanofilaments' to distinguish them from the classic actin-based microfilaments, intermediate filaments, and microtubules of larger diameter [1, 2, 9, 29, 30]. With regard to the cytoarchitecture of algal cells, centrin is assembled into a variety of specialized organelles that include the nucleus basal body connector (striated flagellar root), distal fiber, paraxonemal fiber, fibrous bundle, and transition zone. Finally, each of these organelles is characterized by calcium-modulated supercoiling of its constituent centrinbased filaments and contractile behavior. The centrosome of metazoan cell types is the functional, structural, and molecular homolog of the algal flagellar basal body apparatus [6, 21, 40]. Both organelle complexes function as division centers and microtubule organizing centers. Structurally, the centrosome, flagellar apparatus, and spindle poles harbor basal bodies or centrioles, microtubules, and pericentriolar material [8, 12, 23, 39]. Pericentriolar material may take the form of a fi-

brous reticulum (pericentriolar matrix), or it may be organized into well-formed filamentous organelles such as basal feet, striated roots, distal and proximal connecting fibers, alar fibers, and pericentriolar satellites. Immunolocalization studies at electron microscopic resolution demonstrate that pericentriolar matrix, basal feet, and pericentriolar satellites of metazoan cells label with anticentrin antibodies [4, 26]. Together, these elements constitute a fibrous centrosomal network called the pericentriolar lattice [3-7]. We present evidence that the pericentriolar lattice of metazoan cells is, in part, composed of centrin.

Materials and m e t h o d s

CeH culture PtK 2 rat kangaroo kidney epithelial ceils ([38]; American Type Culture Collection, Rockville, MD) were maintained in minimum essential medium at 37°C and 5% CO 2 in air. Minimum essential medium was supplemented with 10~/0 fetal bovine serum, l mM sodium pyruvate, and 2 mM L-glutamine and buffered to pH 7.3 with 20 mM (N-[2-hydroxyethyl]piperazine-N'[2-ethanesulfonic acid]). QT6 quail fibroblasts ([25]; American Type Culture Collection, Rockville, MD) were grown in NaHCO 3 buffered Dulbecco's modified Eagle medium (pH 7.3), at 37°C and 5°70 CO 2 in air. Dulbecco's modified Eagle medium was supplemented with 4% heat inactivated fetal bovine serum, I% chicken serum, and l mM sodium pyruvate. All tissue culture reagents were purchased from Gibco Laboratories (Grand Island, NY).

Antibody reagents The generation and characterization of anticentrin antiserum, 08/28, and monoclonal anticalmodulin antibody, M/19(6D4), have been described previously [16, 36]. Briefly, antiserum 08/28

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was prepared against electrophoretically purified centrin from isolated striated flagellar roots of the alga Tetraselmis striata. Monoclonal antibody M/19(6D4) was generated against chromatographically purifed Dictyostelium discoideum calmodulin coupled to keyhole limpet hemacyanin. To generate anticentrin antisera 26/14-1 and 26/14-2, a cDNA clone encoding Chlamydomonas reinhardtii centrin was obtained by screening a 2 gtl 1 cDNA library (obtained from Dr S Gantt, Department of Botany, University of Minnesota) with oligonucleotide probes derived from primary sequence analysis of algal centrin (caltractin) [15]. Three positive partial-length clones were identified and a full-length 1049 base pair cDNA encoding the entire centrin protein was constructed. Restriction mapping and sequence analysis demonstrate that this cDNA contains a single initiation codon (ATG) 44 base pairs from its 5' end and an open reading frame of 510 bases. The open reading frame is predicted to encode a protein of 169 amino acids with a calculated molecular mass of 19459 kDa. For antigen production, the centrin cDNA was ligated into the pATH-2 expression vector [1 l] and used to transform E coil RR1. Ampicillin-resistant colonies were selected and screened for expression of a predicted M~ of 57000 trpE-centrin fusion protein. A positive clone, designated ptrpCEN, was grown in M9 media and overexpressed with indoleacrylic acid. Whole cell lysates containing trpE-centrin were prepared and gel purified as described previously [36]. Polyclonal rabbit antisera 26/14-1 and 26/14-2 were generated against purified trpE-centrin by Cocalico Biologicals, Inc (Reamstown, PA).

- 7 0 ° C , and developed with a Kodak M35A X-OMAT film processor (Eastman Kodak Co, Rochester, NY).

Western blotting and 4SCa autoracliography For Western blotting, we used a modified version of the protocol designed specifically for calmodulin [16]. Proteins were transferred to Immobilon polyvinylidene difluoride (PVDF) membrane (Millipore Corporation, Bedford, MA) in modified Towbin transfer buffer [28]. We substituted a solution of 507o non-fat dry milk, l0 mM Tris-HCI (pH 7.4), 150 mM NaCI, 1 mM CaCI 2, and 0.0407o sodium azide as blocking buffer. All antibody reagents were diluted in RPMI 1640 medium containing 2007o fetal bovine serum. Protein visualization was accomplished with alkaline phosphatase-conjugated antibodies (l: 1000 dilution; Jackson ImmunoResearch Laboratories, Inc PA) and bromochloroindolyl phosphate/nitro blue tetrazolium as substrate [13]. 45Ca autoradiography of proteins transferred to PVDF membrane was used to detect calcium-binding proteins [20]; the membrane was subsequently Western blotted with 26/14-1 and M/19(6D4) to detect centrin and calmodulin, respectively.

Immunofluorescence microscopy Cells were processed for immunofluorescence microscopy as previously described [3] and photographed with Hypertech film (Microfluor, Ltd, Stony Brook, NY).

Electrophoresis SDS-PAGE was carried out in slab gels containing a 3-20°70 gradient of acrylamide [17]. First-dimension isoelectric focusing tube gels were run with 1.6070Biolyte 4 - 6 and 0.4070 Biolyte 3-10 (BioRad Laboratories, Richmond, CA), and 407o total acrylamide [27]. The second dimension SDS-PAGE was run in slab gels containing 15°70 acrylamide.

Immunoprecipitation Immtmoprecipitation of PtK 2 and QT6 whole cell lysates was performed as described by Baron and Salisbury [4] with several modifications. Immunoprecipitates were analyzed by either Western blotting or autoradiography. For autoradiography, cells were preincubated in Dulbecco's modified Eagle medium minus methionine and cysteine, labeled with Dulbecco's modified Eagle medium containing [35S]methionine and [35S]cysteine (50 tzCi/ml each; Amersham Corp, Arlington Heights, IL), rinsed with PBS, trypsinized, harvested by centrifugation, washed with PBS containing 0.15 mg/ml phenylmethyl sulfonyl fluoride, and lysed as previously described. Approximately 2.5 x 107 or 2.5 × 106 cells were used for each unlabeled or [3SS]labeled gel sample, respectively. Cell lysates were immediately diluted with cold (0-4°C) immunoprecipitation buffer A (50 mM Tris-HCl (pH 7.4), 190 mM NaCl, 1 mM CaCl 2, 2.5070 Triton X-100) containing protease inhibitors (0.15 mg/ml phenylmethyl sulfonyi fluoride, 2.0/zg/ml aprotinin, 1.0/zg/ml leupeptin, 2.0/zg/ml pepstatin A), incubated with the appropriate primary antisera (1:250 dilution) at 0 - 4 ° C overnight, and incubated with protein A/G-Sepharose (Piere, Rockford, IL) for 1-2 h at 0 - 4 ° C . Protease inhibitors were added to immunoprecipitation buffer A just prior to use. All cell lysates were incubated with the equivalent of a 20-~1 packed protein A/G-Sepharose pellet per gel lane. Protein A/G-Sepharose pellets were washed with three changes of cold immunoprecipitation buffer B (10 mM Tris-HCl (pH 8.3), 150 mM NaCl, 1 mM CaCI 2, 0.1070 Triton X-100) followed by three changes ofTBS (10 mM Tris-HCl, 150 mM NaCl, pH 7.4). Final protein A/G-Sepharose pellets were resuspended with 50 tzl 2 x Laemmli sample buffer or isoelecta'ic focusing sample buffer and subjected to electrophoresis. Gels containing radioactive samples were impregnated with Pro-Mote fluorography enhancer (Integrated Separation Systems, MA) according to the manufacturers instructions, dried, recorded on Kodak XAR' film at

Results

Immunofluorescence microscopy I m m u n o f l u o r e s c e n c e micrographs o f interphase rat kang a r o o kidney epithelial PtK 2 cells and quail QT6 fibroblasts double-labeled with antitubulin and anticentrin antibodies identify the pericentriolar lattice as a constellation o f pericentrosomal spots (fig 1). All o f our anticentrin antibodies give an indistinguishable localization pattern. In P t K 2 cells these spots are n u m e r o u s and distributed some distance f r o m the point o f microtubule focus (fig 1B), whereas in QT6 cells they are fewer in number and closely associated with the centriolar region (fig 1D). Baron et al [3, 5] have d e m o n s t r a t e d that the distribution pattern o f pericentrosomal spots in P t K 2 cells changes in a cell cycle dependent manner. In mitotic cells, centrin is a c o m p o n e n t o f the spindle poles and spindle apparatus (fig 2). Numerous pericentrosomal spots can be seen at the poles and within the spindle o f metaphase P t K 2 cells (fig 2 B , B ' , B " ) . As in interphase QT6 cells, the distribution o f spots in mitotic QT6 cells is closely associated with the centriolar region (fig 2E,E'). I m m u n o g o l d localization studies have c o n f i r m e d t h a t the a n t i c e n t r i n immunofluorescence labeling pattern o f pericentrosomal spots corresponds to pericentriolar matrix, basal feet, and pericentriolar satellites at electron microscopic resolution [4, 26].

Immunoprecipitation, Western blot, and 45Ca autoradiography We have previously identified a protein o f M r 165000 in [3SS]labeled whole cell lysates o f P t K 2 cells by immunoprecipitation with anticentrin polyclonal antiserum 08/28 [4]. Our laboratory has since generated two new rabbit polyclonal antibodies toward a trpE-centrin fusion protein, designated 26/14-1 and 26/14-2. All three antibody'

Characterization of pericentriolar lattice

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t Fig 1. lmmunofluorescence micrographs of an interphase PtK 2 (A,B) and QT6 (C,D) cell double-labeled with antitubulin (A,C) and anticentrin (B,D) antibodies are shown. The PtK 2 and QT6 cells were labeled with anticentrin antiserum 08/28 and 26/14-1, respectively. The pericentriolar lattice is seen as a constellation of pericentrosomal spots. In PtK 2 cells (B) these spots (white arrowheads) are more numerous and distributed some distance from the point of microtubule focus, whereas in QT6 cells (D) they are fewer in number and closely associated with the centriolar region. Bar, 10 #m.

Fig 2. Immunofluorescence micrographs of a metaphase PtK 2 ( A - C ) and QT6 ( D - F ) cell triple-labeled with antitubulin (A,D) and anticentrin (B,B',B",E,E') antibodies, and the DNA-binding fluorochrome DAPI (C,F) are shown. The PtK 2 and QT6 cells were labeled with anticentrin antiserum 08/28 and 26/14-1, respectively. The chromosomes in both cells are aligned at the, cell equator (C,F). Several focal planes of anticentrin-labeling are shown for both cells (B,B',B",E,E'). The spindle poles (open white arrows) are demarcated by clusters of pericentrosomal spots. Additional spots (white arrowheads) are found within the PtK 2 spindle, extending from the poles to the chromosomes. In contrast, anticentrin-labeling in the QT6 cell is primarily confined to the spindle poles. Bar, l0 ¢m.

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D Fig 3. Autoradiograms of [3SS]labeled PtK 2 (A,C) and QT6 (B,D) Immunoprecipitates are shown. A,B. Whole cell lysates were immuno19reci19itated with 08/28 immune serum (a,a'), 08/28 19reimmune serum (b,b'), 26/14-I immune serum (c,c'), 26/14-1 19reimmune serum (d,d'), 26/14-2 immune serum (e,e'), and 26/14-2 19reimmune serum (f,f'). Proteins of M r 165000 and 64000 (arrows) and several low molecular mass centrins (brackets) are immuno19reci19itated specifically. Molecular mass markers (in kDa) indicated by the ticks, from top to bottom, are 205000 (myosin); 116000 ~-galactosidase); 97400 (19hos19horylase B); 66000 (bovine serum albumin); 45000 (albumin, egg); 36000 (glyceraldehyde-3-19hos19hate dehydrogenase); 29000 (carbonic anhydrase); 24000 (try19sinogen); 20 I00 (soybean trypsin inhibitor); and 14200 (a-lactalbumin). C,D. Whole cell lysates of PtK, and QT6 cells were immuno19reci19itated with 26/14-1 immune serum. Proteins were separated electrophoretically by isoelectric focusing in the first dimension and SDSPAGE in the second dimension. Several centrin isoforms with acidic 19Is(4.4-5.4) are resolved. The acidic region of the gel is printed toward the right. Isoelectric focusing standards (in kDa) indicated by the asterisk are as follows: 20100; pl 4.55 (soybean trypsin inhibitor); 18400; 191 5.15 (/3-1actoglobulin).

reagents recognize centrin from various species of algae by Western blot analysis (.not shown), and immunoprecipitate the M r 165000 protein from [35S]labeled whole cell lysates of PtK 2 cells (fig 3A). In addition, 26/14-2 forms an antigen-antibody complex with a set of low molecular mass proteins of M r 18 500, 19 500 and 20000. Antiserum 26/14-1 also precipitates these low molecular mass proteins plus an additional band of M r 21000, which comigrates with algal centrin (not shown). Two-dimensional gel analysis of the low molecular mass proteins immunoprecipitated with 26/14-1 resolves six distinct isoforms with acidic pls between 4.4 and 5.4 (fig 3C). Immunoprecipitation of [35S]labeled whole cell lysates of QT6 fibroblasts with 08/28, 26/14-1, and 26/14-2 demonstrates that these cells express a similar set of proteins (fig 3B,D). Although 08/28 does not immunoprecipitate any specific proteins from QT6 cells, both 26/14-1 and 26/14-2 react with a closely spaced doublet of M r 19500/20000. Two-dimensional gel electrophoresis shows that the M r 19500 and 20000 proteins of QT6 cells have acidic pls of 5.0 and 5.4, respectively (fig 3D). Ifi addition, 26/14-1 and 26/14-2 form an immune complex with proteins of M r 165 000 and 64000, respectively. These results suggest that the M r 165000-, 64000-, and 18500-21000 profeins of

PtK 2 and QT6 cells share an immunologic epitope(s) with algal centrin. Alternatively, some of these molecules may be part of a multi-protein complex that immunoprecipitates with the centrin epitope(s). Analysis of 26/14-1 immune complexes from PtK 2 and QT6 cells by Western blot and 45Ca autoradiography demonstrates that at least one protein of approximately M r 20000, from both cell types, binds calcium (fig 4). Note that the M r 21000 protein of PtK 2 cells is not labeled by Western blotting in this experiment. Our standard Western blotting protocol includes glutaraldehyde fixation of the proteins to the P V D F membrane ; this step is omitted for 45Ca overlay autoradiography. Because we can routinely resolve and Western blot each of the low molecular mass PtK 2 and QT6 isoforms with anticentrin antisera in slab gels containing 15~0 acrylamide (not shown), we believe that the M r 21000 protein has been washed from the membrane and is no longer present in an amount detectable by our Western blotting protocol. We have performed the 45Ca overlay experiment with glutaraldehyde fixation, and found that only calmodulin retains its ability to bind calcium after fixation. At the present time, we have not observed labeling of either the M r 64000 or 165000 proteins by Western blot or 45Ca

Characterization of pericentriolar lattice

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Fig 4. A. Western blot and (B) 45Ca autoradiogram of purified bovine brain calmodulin (Sigma Chemical Company, St Louis, MO) (a,a'), and PtK 2 centrins (b,b') and QT6 centrins (c,c') immunoprecipitated with 26/14-1 are shown. Both calmodulin and at least one centrin isoform of approximately M, 20000 from the PtK 2 and QT6 immunoprecipitates bind 45Ca after SDSPAGE and electrotransfer to PVDF membrane. The membrane was first processed for 45Ca autoradiography and then subsequently Western blotted with 26/14-1 and M/19(6D4) to detect centrin and calmodulin, respectively. Molecular mass markers (in kDa) indicated by the ticks, from top to bottom, are 66000 (bovine serum albumin); 45000 (albumin, egg); 36000 (glyceraldehyde-3-phosphate dehydrogenase); 29000 (carbonic anhydrase); 24000 (trypsinogen); 20100 (soybean trypsin inhibitor); and 14200 (a-lactalbumin).

overlay autoradiography. Taken together, the molecular mass, acidic p/s, calcium-binding properties, and immunoreactivity of the low molecular mass PtK 2 and QT6 proteins with anticentrin antibodies indicate that they are centrin isoforms of metazoan cells.

Discussion Centrin was first identified by Salisbury and coworkers [36] in the green alga Tetraselmis striata, where it was shown to be the major protein component of,partially purified striated flagellar roots. At that time, our laboratory generated antiserum 08/28 and confirmed the localization of centrin to striated flagellar roots by immunocytochemistry. Electrophoretic analysis showed centrin to be a low molecular mass protein of approximately M r 21000 with two acidic isoforms, ct (pI = 4.9) and fl (pI = 4.8). Metabolic labeling with 32po 4 further reveaied that fl-centrin is a phosphorylated form of a-centrin.

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More recently, another phosphorylated isoform, 7-centrin (pI = 4.7), has been identified in Tetraselmis striata [l 9]. Immunolocalization studies with anticentrin antibody 08/28 subsequently demonstrated the association of a centrin epitope(s) with the flagellar basal body apparatus and centrosome of many algal and mammalian cell types, respectively [3 l, 37]. For all algal cells examined to date, Western blot analysis with 08/28 has consistently identified a molecule of approximately M r 20000 to 21000 [10, 14, 18, 22, 35, 37, 41]. The situation for metazoan cells, however, has been complicated by the identification of a M r 165000 protein in PtK 2 rat kangaroo cells by immunoprecipitation of [35S]labeled whole cell lysates [4] and a M r 62000/64000 protein doublet in KE37 h u m a n lymphoblasts by Western blot of isolated centrosorn'es [26] with antibody 08/28. Here, we show that two newlydeveloped anticentrin polyclonal antisera (26/14-1 and 26/14-2) form immune complexes with a set of acidic low molecular mass calcium-binding proteins in PtK 2 (M r 18500 through 21000) and QT6 (M r 19500/20000 doublet) cells. Although antiserum 08/28 does not precipitate these low molecular mass proteins, it does react with a subset of them on Western blots after they have been immunoprecipitated with 26/14-1 (Baron and Salisbury, unpublished observations). In addition to the low molecular mass proteins, antisera 26/14-1 and 26/14-2 imm u n o p r e c i p i t a t e the M r 165000 a n d / o r M r 64000 proteins from QT6 and PtK 2 cells. We have tried to Western blot the M r 165000 protein, only to find that it is inefficiently transferred from the gel (Baron and Salisbury, unpublished observations). We have also not observed labeling of the M r 64000 protein by Western blot. We can now make several statements regarding the pericentriolar lattice of metazoan cells and its protein composition, l) PtK 2 and QT6 cells each express a unique set of low molecular mass proteins, which, based on their molecular masses, acidic p/s, calcium-binding properties, and immunoreactivity with several anticentrin antibodies are centrin isoforms. Multiple protein isoforms may result from multiple copies of a gene, from alternate transcripts of one gene, and from post-translational modification o f the nascent protein. 2) The M r 165000 protein of QT6 and PtK 2 cells, the M r 64000 protein of QT6 cells, and the M r 62000/64000 doublet of KE37 cells share an epitope(s) with centrin. Although it is possible that the M r 165000 protein is a centrin-binding protein that coprecipitates with centrin, the observation that 08/28 precipitates this protein from PtK 2 cells without any detectable centrin argues against this hypothesis (see fig 3A). We have seen no labeling of the M r 64000 protein of QT6 cells with our Western blot methods, but Moudjou and coworkers [26] have Western blotted a M r 62000/64000 doublet from isolated centrosomes of human iymphoblasts with antiserum 08/28. Although it is possible that the M r 64000 protein of QT6 cells is a centrin-binding protein that co-precipitates with centrin, the observation that 08/28 Western blots a protein doublet of similar molecular mass from lymphoblasts also argues against this hypothesis. At the present time, the molecular basis for this common centrin epitope(s) remains unclear. The M r 165000, 64000, and 62000/64000 proteins could be the products of tandem gene duplication, gene fusion with other genes, a n d / o r alternate slicing of mRNAs. 3) The pericentriolar lattice as defined by anticentrin antisera is, in part, composed of the M r 64000 and 165000 centrin-related proteins, and centrin. Careful biochemical and molecular analysis are now necessary to dissect the structural and

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functional relatedness o f different centrin isoforms and centrin-related proteins, as well as the functional and structural contributions they make to the pericentriolar lattice.

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Acknowledgments

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We thank Mr Mark Sanders and Mr Tien Danh for photographic assistance. This work was supported by NIH grant GM 35258 to JLS and NIH training grant CA 09441 to ATB.

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References

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