Saturable binding of the echinoderm microtubule-associated protein (EMAP) on microtubules, but not filamentous actin or vimentin filaments.

The echinoderm microtubule-associated protein (EMAP) is a 75-kDa, WD-repeat protein associated with the mitotic spindle apparatus. To understand EMAP's biological role, it is important to determine its affinity for microtubules (MTs) and other cytoskeletal components. To accomplish this goal, we utilized a low-cost, bubble-column bioreactor to express EMAP as a hexahistidine fusion (6his) protein in baculovirus-infected insect cells. After optimizing cell growth conditions, up to 30 mg of EMAP was obtained in the soluble cell lysate from a 1-liter culture. EMAP was purified to homogeneity in a two-step process that included immobilized metal-affinity chromatography (IMAC) and anion-exchange chromatography. In vitro binding studies on cytoskeletal components were performed with the 6his-EMAP. EMAP bound to MTs, but not actin or vimentin filaments, with an intrinsic dissociation constant of 0.18 microM and binding stoichiometry of 0.7 mol EMAP per mol tubulin heterodimer. In addition, we show that a strong MT binding domain resides in the 137 amino acid, NH(2)-terminus of EMAP and a weaker binding site in the WD-domain. Previous work has shown that the EMAP concentration in the sea urchin egg is over 4 microM. Together, these results show that there is sufficient EMAP in the egg to regulate the assembly of a large pool of maternally stored tubulin.

Conservation of the WD-repeat, microtubule-binding protein, EMAP, in sea urchins, humans, and the nematode C. elegans.

The echinoderm microtubule-associated protein (EMAP) is the most abundant microtubule-binding protein in the first cleavage mitotic apparatus in sea urchin embryos. The first goal of this study was to determine whether there is sufficient EMAP in the egg and embryo to modify microtubule dynamics during the early cleavages divisions and whether EMAP functions at a specific time or place in the embryo. To accomplish this goal, we examined the relative abundance, tissue distribution, and temporal pattern of EMAP expression during embryonic development. The second goal of this study was to identify important functional domains within the EMAP coding sequence. A conserved sequence might reveal a potential microtubule-binding domain. We cloned, sequenced and compared overlapping EMAP cDNAs from two different sea urchin species that diverged approximately 80 million years ago, and compared these with cDNA sequences from a vertebrate and nematode species. From quantitative immunoblots, we determined the EMAP concentration in eggs to be 4 microM. The steady-state levels of EMAP mRNA and protein accumulated during development, and all three germ layers expressed EMAP. During the early stages of development, EMAP and tubulin were both abundant in the ectoderm, mesoderm and endoderm. However, during late gastrulation and the formation of the early pluteus larvae, EMAP was enriched in the mesoderm, while tubulin staining was most abundant in the archenteron. These results indicate that EMAP may have tissue-specific functions in the late stage embryo. To identify conserved functional domains, we compared the predicted amino acid sequence encoded by Strongylocentrotus purpuratus and Lytechinus variegatus EMAP cDNAs, and determined that these two sea urchin EMAPs were 95% conserved and shared an identical domain organization. A parsimonious analysis of these sea urchin protein sequences, as well as human and C. elegans EMAP sequences was used to construct a gene tree. Together these results suggest that EMAP is an important microtubule protein required at all developmental stages of sea urchins, and whose cellular function may be conserved amongst metazoans.

Sequence and expression patterns of a human EMAP-related protein-2 (HuEMAP-2).

Until recently, the microtubule-associated protein, EMAP, was identified only in echinoderms such as sea urchin, starfish and sand dollar. Sea urchin EMAP localizes to the mitotic apparatus in vivo and modifies the assembly dynamics of microtubules in vitro. To identify domains important for EMAP function, we cloned and sequenced cDNAs for an EMAP-related protein in human. The nucleotide sequence of a human EMAP-related protein-2 (HuEMAP-2) encodes a protein of 649 amino acids in length. The translated polypeptide sequence and domain structure of sea urchin EMAP and HuEMAP-2 are highly conserved, with greater than 57% identity and 77% similarity at the translated amino acid level. Southern blot analysis is consistent with the presence of a single HuEMAP-2 gene in the human genome. Moreover, HuEMAP-2 is a member of a larger protein family with at least four HuEMAP sequences in the NCBI databases. One of these, HuEMAP-1, is identified as the candidate gene for the Usher syndrome 1 a locus (Genomics 43:104-106, 1997). Northern blot analysis indicates that HuEMAP-1, and HuEMAP-2 are expressed in different human tissues. In addition, these RNA blots indicate that HuEMAP-2 transcripts may be differentially spliced in neuronal tissues.

The 77-kDa echinoderm microtubule-associated protein (EMAP) shares epitopes with the mammalian brain MAPs, MAP-2 and tau.

Previous work has shown that the echinoderm microtubule-associated protein (EMAP) was a unique MAP with little sequence similarity with the brain MAPs. The purpose of this study was to determine whether there were any small domains within EMAP that were shared by the mammalian brain MAPs, MAP-2, and tau. It is reported here that EMAP and the heat-stable MAP-2 and tau share antigenic determinants. A polyclonal antisera, raised against SDS-PAGE denatured EMAP, reacted strongly with both MAP-2 and tau on Western blots. In addition, a detailed sequence comparison, using a window of 5 amino acids at a time, revealed several short domains with approximately 20 residues that shared sequence similarity. The regions of sequence similarity were all located in regions implicated in microtubule binding, suggesting that EMAP and the mammalian brain MAPs may share short structural and functional domains.

Overexpression of the 77-kD echinoderm microtubule-associated protein (EMAP), a WD-40 repeat protein, in baculovirus-infected Sf9 cells.

The purpose of this study was to test whether any assembly-promoting microtubule-associated protein (MAP) would bundle microtubules and induce process formation in recombinant baculovirus-infected Sf9 cells, in particular, whether a non-neural MAP from a normally rounded cell would produce cellular asymmetries. To carry out these experiments, we constructed a recombinant baculovirus that expressed the full-length 77-kD EMAP, an abundant MAP that localizes to the mitotic spindle of cleavage-stage sea urchin embryos and to the interphase array of microtubules in adult coelomocytes. Expression of EMAP in Sf9 cells had no detectable effect on cellular morphology, microtubule organization, or stability. These results indicate that process formation in Sf9 cells is MAP specific.

Purification of a WD repeat protein, EMAP, that promotes microtubule dynamics through an inhibition of rescue.

The major microtubule-associated protein in echinoderms is a 77-kDa, WD repeat protein, called EMAP. EMAP-related proteins have been identified in sea urchins, starfish, sanddollars, and humans. We describe the purification of sea urchin EMAP and demonstrate that EMAP binding to microtubules is saturable at a molar ratio of 1 mol of EMAP to 3 mol of tubulin dimer. Unlike MAP-2, MAP-4, or tau proteins, EMAP binding to microtubules is not lost by cleavage of tubulin with subtilisin. In addition to binding to the microtubule polymer, EMAP binds to tubulin dimers in a 1:1 molar ratio. The abundance of EMAP in the egg suggests that it could function to regulate microtubule assembly. To test this hypothesis, we examined the effects of EMAP on the dynamic instability of microtubules nucleated from axoneme fragments as monitored by video-enhanced differential interference contrast microscopy. Addition of 2.2 microM EMAP to 21 microM tubulin results in a slight increase in the elongation and shortening velocities at the microtubule plus ends but not at the minus ends. Significantly, EMAP inhibits the frequency of rescue 8-fold without producing a change in the frequency of catastrophe. These results indicate that EMAP, unlike brain microtubule-associated proteins, promotes microtubule dynamics.

Characterization of the sea urchin major vault protein: a possible role for vault ribonucleoprotein particles in nucleocytoplasmic transport.

Vaults are large ribonucleoprotein particles that have been identified in a wide range of eukaryotic organisms. Although present in thousands of copies per cell, their function remains unknown. In this report, we identify the major vault protein in sea urchins as a 107-kDa polypeptide that copurifies with microtubules and ribosomes. Although initially identified in microtubule preparations, the sea urchin major vault protein is not predominantly microtubule-associated in vivo. Rather, the sea urchin major vault protein is present throughout the cytoplasm in eggs and embryos and in the nucleus in adult somatic cells. Within the nucleus, the sea urchin major vault protein is concentrated in the region of the nucleolus and to punctate regions of the nuclear envelope. In addition, the vault protein localizes to short linear strings juxtaposed to the exterior of the nucleus and extending outward into the cytoplasm. Based on their copurification and intracellular distribution, vaults may be involved in the nucleocytoplasmic transport of ribosomes and/or mRNA.

Cell cycle-dependent phosphorylation of the 77 kDa echinoderm microtubule-associated protein (EMAP) in vivo and association with the p34cdc2 kinase.

The most abundant microtubule-associated protein in sea urchin eggs and embryos is the 77 kDa echinoderm microtubule-associated protein (EMAP). EMAP localizes to the mitotic spindle as well as the interphase microtubule array and is a likely target for a cell cycle-activated kinase. To determine if EMAP is phosphorylated in vivo, sea urchin eggs and embryos were metabolically labeled with 32PO4 and a monospecific antiserum was used to immunoprecipitate EMAP from 32P-labeled eggs and embryos. In this study, we demonstrate that the 77 kDa EMAP is phosphorylated in vivo by two distinct mechanisms. In the unfertilized egg, EMAP is constitutively phosphorylated on at least five serine residues. During the first cleavage division following fertilization, EMAP is phosphorylated with a cell cycle-dependent time course. As the embryo enters mitosis, EMAP phosphorylation increases, and as the embryo exits mitosis, phosphorylation decreases. During mitosis, EMAP is phosphorylated on 10 serine residues and two-dimensional phosphopeptide mapping reveals a mitosis-specific site of phosphorylation. At all stages of the cell cycle, a 33 kDa polypeptide copurifies with the 77 kDa EMAP, regardless of phosphorylation state. Antibodies against the cdc2 kinase were used to demonstrate that the 33 kDa polypeptide is the p34cdc2 kinase. The p34cdc2 kinase copurifies with the mitotic apparatus and immunostaining indicates that the p34cdc2 kinase is concentrated at the spindle poles. Models for the interaction of the p34cdc2 kinase and the 77 kDa EMAP are presented.

Molecular characterization of the 77-kDa echinoderm microtubule-associated protein. Homology to the beta-transducin family.

The major microtubule-associated protein (MAP) of sea urchins and several other echinoderms is a polypeptide of M(r) 77,000. The echinoderm MAP (EMAP) is abundant in embryonic and differentiated cells, as well as in mitotic and interphase microtubule arrays. To characterize the molecular structure and function of the EMAP, we isolated a full-length cDNA clone, which has one open reading frame that predicts a polypeptide of 686 amino acids with a calculated M(r) of 75,488. On the basis of charge distribution, EMAP can be divided into two distinctive domains: The NH2-terminal basic region (amino acids 1-137, pI = 10.0) and a slightly acidic, COOH-terminal region (amino acids 138-686, pI = 5.8). This charge distribution is typical of many microtubule-binding proteins, but no significant sequence homology has been detected with any known MAPs. The EMAP, however, does show significant sequence similarity with the beta-subunit of the heterotrimeric G-protein, transducin. The homology lies in a series of 10 imperfect, 43-amino acid repeats (WD-40 repeats) that have been found in many proteins of diverse functions, including beta-transducins, Drosophila Enhancer of split, the yeast STE4, CDC4, CDC20, PRP4, and Tup1 gene products, and the dTAFII80 subunit of Drosophila TFIID. The function of these repeats still remains unknown. It is possible that these repeats are involved in protein-protein interactions, perhaps with the tetratricopeptide repeat-containing protein family. Alternatively, the EMAP may be an important link between signal transduction events and a change in microtubule organization during the cell cycle.

Polyribosome targeting to microtubules: enrichment of specific mRNAs in a reconstituted microtubule preparation from sea urchin embryos.

A subset of mRNAs, polyribosomes, and poly(A)-binding proteins copurify with microtubules from sea urchin embryos. Several lines of evidence indicate that the interaction of microtubules with ribosomes is specific: a distinct stalk-like structure appears to mediate their association; ribosomes bind to microtubules with a constant stoichiometry through several purification cycles; and the presence of ribosomes in these preparations depends on the presence of intact microtubules. Five specific mRNAs are enriched with the microtubule-bound ribosomes, indicating that translation of specific proteins may occur on the microtubule scaffolding in vivo.

pH-dependent solubility and assembly of microtubules in bovine brain extracts.

Alkaline pH favors the assembly of microtubules (MTs) in marine egg extracts [Suprenant and Marsh, 1987: J. Cell Sci. 184:167-180; Suprenant, 1989: Exp. Cell Res. 184:167-180; 1991: Cell Motil. Cytoskeleton 19:207-220] and mammalian brain extracts [Tiwari and Suprenant, 1993: Anal. Biochem. 215:96-103], even though the assembly of purified microtubule protein (MTP) from both of these sources is favored at slightly acidic pH. The present investigation examines whether alkaline pH has a direct or indirect effect on MT nucleation and growth in soluble brain extracts. Cell-free extracts were prepared from bovine cerebral cortex, and a nucleated assembly assay was used to demonstrate that MT assembly in brain extracts is favored at slightly acidic pH. The increase in MT mass found at alkaline pH is due to an increase in the solubility of tubulin not an increase in the extent of assembly. On average, 47.7 +/- 11.3% of the total tubulin is soluble at pH 7.2, while only 30.9 +/- 8.9% of the tubulin is soluble at pH 6.8. A model is proposed that indicates how microtubule proteins from both mammalian brain and marine eggs may be associated with pH-dependent factors.

A pH- and temperature-dependent cycling method that doubles the yield of microtubule protein.

This report describes a procedure for the isolation of microtubules (MTs) and microtubule-associated proteins from mammalian cultured cells and tissues. This method relies on the discovery that the solubility of brain tubulin is pH-sensitive. In this report, we examined tubulin solubility over a broad pH range and discovered that the amount of soluble tubulin increased by 240% as the pH was raised from 6.0 to 8.0. Quantitative immunoblotting revealed that there was an almost equal partitioning of tyrosinated and acetylated alpha-tubulin isotypes at the higher pH. These observations were incorporated into a new procedure for the purification of microtubule protein (MTP) from bovine brain and mouse B16 cultured melanoma cells. Morphologically, and in terms of polypeptide composition, this MTP is indistinguishable from that prepared by a glycerol-cycling method (21). Moreover, our new method of pH- and temperature-cycling yields almost twice as much MTP as that obtained by other cycling methods.

EMAP, an echinoderm microtubule-associated protein found in microtubule-ribosome complexes.

The major non-tubulin polypeptide found associated with microtubules purified from unfertilized sea urchin eggs by cycles of pH-dependent assembly has a Mr of 77,000. The 77,000 Mr polypeptide is heat- and acid-labile, and is antigenically distinct from the mammalian brain MAPs, MAP-2 and tau. Affinity-purified antiserum against the 77,000 Mr polypeptide was used to survey a variety of cells and tissues for the presence of antigenically related polypeptides. A cross-reacting polypeptide, ranging in Mr from 72,000 to 80,000, was found in microtubule preparations from a wide variety of echinoderms, including sea urchins, starfish and sand dollars. Indirect immunofluorescence showed that the polypetide was found in interphase as well as mitotic microtubule arrays. No cross-reacting material was detected in microtubules isolated from marine molluscs, mammalian brain or mouse B16 cultured cells. Because the 77,000 Mr MAP is abundant in echinoderms, we have called it EMAP for echinoderm microtubule-associated protein. Although the precise function of the EMAP is not known, our data suggest that the EMAP is involved in the attachment of ribosomes to microtubules. Large numbers of ribosomes are attached to the walls of EMAP-containing microtubules, but not EMAP-deficient microtubules. Removal of the EMAP from the microtubule by salt-extraction results in the release of ribosomes from the microtubule, indicating that the EMAP may form part or all of the long tapered stalk that connects these two organelles.

Particulate Tubulin in Interphase and Metaphase Extracts of Oocytes of Spisula.

In 1972 Weisenberg reported that surf clam oocytes contained a particulate and sedimentable pool of tubulin that could be isolated in buffers containing hexylene glycol. This "interphase particulate tubulin" (IPT) copurified with 10-20 {mu}m granular spheres, which were identified as the "tubulin-containing structures" (TCS). Approximately one TCS per oocyte was isolated and the TCS disappeared after nuclear envelope breakdown. Weisenberg postulated that the TCS was comprised of a stored form of tubulin or a microtubule-assembly intermediate. To characterize this intriguing form of stored tubulin, IPT was isolated in hexylene glycol-containing buffers as described by Weisenberg (1972, J. Cell Biol. 54, 266-278) and the amount of sedimentable tubulin was quantitated by immunoblotting during the first meiotic cell cycle. Approximately 10% of the total tubulin in Spisula oocytes sediments at g forces that are too small to pellet tubulin dimers or even single microtubules. Granular spheres, approximately 15 {mu}m in diameter, are present in the sedimentable tubulin fractions. During the first cell cycle, the granular spheres disappear while the sedimentable tubulin levels gradually decrease. Although the disappearance of the spheres corresponds with the loss of sedimentable tubulin, the spheres do not contain tubulin. An initial centrifugation of the oocyte homogenates at 650g leaves most of the tubulin in the supernatant and the granular spheres in the pellet. The tubulin-containing fractions are composed of membranes and an amorphous unidentified material associated with short microtubules. Sedimentable tubulin is not detected in homogenates prepared at 0{deg}C or in the absence of hexylene glycol, conditions that favor microtubule disassembly. It is likely that sedimentable tubulin is composed of hexylene glycol-induced polymers and not unique particulate structures that sequester tubulin. Finally, the granular spheres that contaminate the tubulin preparations are identified as nucleoli. They are morphologically identical to the nucleoli of the intact oocyte and they fluoresce brightly when stained with the Hoechst DNA dye 22358.

Unidirectional microtubule assembly in cell-free extracts of Spisula solidissima oocytes is regulated by subtle changes in pH.

Most, if not all, microtubules in vivo grow unidirectionally from a nucleation site such as the centrosome. This organized growth of microtubules can generate and maintain the radially symmetrical array of interphase microtubules as well as the bipolar mitotic apparatus. To investigate the regulation of polarized microtubule growth, we have prepared a cell-free extract from surf clam oocytes that exhibits unidirectional microtubule assembly. Immunofluorescence microscopy was used to visualize the net assembly of microtubules onto the fast (plus)- and slow (minus)- growing ends of isolated ciliary axonemes. All detectable microtubule growth in these cytoplasmic extracts occurred at the plus (+) ends and the extent of (+) end growth was regulated by subtle changes in pH. Microtubule assembly in these crude extracts was highly favored at pH 7.3, the pH of the post-fertilization cytoplasm. In contrast, when tubulin was purified from these oocyte extracts, integral components were lost, and microtubule growth became predominantly bidirectional and was favored at acidic pH. These results indicate that cytoplasmic factors may inhibit bidirectional growth in vivo and that temporal or local changes in cytoplasmic pH may influence microtubule assembly during the cell cycle.

Alkaline pH favors microtubule self-assembly in surf clam, Spisula solidissima, oocyte extracts.

Microtubule assembly in surf clam oocytes is dependent upon events that occur during fertilization. Prior to fertilization there are few, if any microtubules, but within minutes after fertilization microtubules assemble to form the meiotic apparatus. This study demonstrates that the assembly of microtubules after fertilization may be dependent on the fertilization-induced pH change of the cytoplasm. Since the magnitude of the intracellular pH (pHi) change in Spisula oocytes has not been determined, surf clam microtubule assembly was examined at pH values that reflect the pHi change that occurs during sea urchin fertilization. The results indicate that microtubule assembly in crude oocyte extracts is favored at alkaline pH. In contrast, purified surf clam tubulin assembles to a greater extent at pH 6.6 than at pH 7.2. These results reveal that the tubulin in unfertilized oocytes can assemble into microtubules at pH 6.6 but that they are prevented from doing so by pH-dependent cytoplasmic regulatory factors in the oocyte.

Association of ribosomes with in vitro assembled microtubules.

Microtubules were purified from unfertilized eggs of the sea urchins Arbacia punctulata, Lytechinus pictus, Lytechinus variegatus, and Strongylocentrotus purpuratus. Numerous densely stained particles (24 x 26 nm) are associated with microtubules isolated from each of these sea urchins. The most striking aspect of this structure is an extended, slightly curved arm that appears to attach the particles to the microtubule. Morphologically similar particles are associated with microtubules of the isolated first cleavage mitotic apparatus. The particles are attached to the microtubules by ionic interactions and contain large amounts of extractable RNA. Based upon their size and density, RNA and protein composition, and sedimentation in sucrose gradients, the microtubule-associated particles are identified as ribosomes.

Release of intact microtubule-capping structures from Tetrahymena cilia.

The distal ends of ciliary microtubules are attached to the membrane by microtubule-capping structures. The capping structures are located at the sites of tubulin addition and loss in vivo and may be part of the regulatory system that directs ciliary and flagellar microtubule assembly. This study describes conditions for the release and stabilization of microtubule capping structures as a first step in their purification. Two types of capping structures, the distal filaments and the central microtubule caps, are selectively and independently released from the axoneme by CaCl2 and MgCl2 but not by MgSO4, ZnCl2, NaCl, KCl, or KI. The release of the caps and filaments is specific for Ca+2, Mg+2, and Cl- and is not simply a function of ionic strength. The capping structures are released without major disruption of the axonemal structure. In addition to providing a means to purify and identify the cap and filament components, these results suggest ways in which their binding to the axoneme may be modulated during periods of microtubule growth or shortening. This report also reveals that the distal filaments are composed of two separable components, a small bead inserted into the end of each A-tubule and a "Y"-shaped plug and filament that slips through the bead.