Polyribosome Targeting to Microtubules

Polyribosome Targeting to Microtubules: Enrichment of Specific mRNAs in a Reconstituted Microtubule Preparation from Sea Urchin Embryos Danielle Hamill,* Jill Davis,* Julie Drawbridge,* and Kathy A. Suprenant* * Department of Physiology and Cell Biology, University of Kansas, Lawrence, Kansas 66045; and *Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544 Abstract. A subset of mRNAs, polyribosomes, and several purification cycles; and the presence of ribo- poly(A)-binding proteins copurify with microtubules somes in these preparations depends on the presence from sea urchin embryos. Several lines of evidence in- of intact microtubules. Five specific mRNAs are en- Downloaded from http://rupress.org/jcb/article-pdf/127/4/973/1264027/973.pdf by guest on 27 September 2021 dicate that the interaction of microtubules with ribo- riched with the microtubule-bound ribosomes, indicat- somes is specific: a distinct stalk-like structure ap- ing that translation of specific proteins may occur on pears to mediate their association; ribosomes bind to the microtubule scaffolding in vivo. microtubules with a constant stoichiometry through N many embryos, the pattern of development that fol- et al., 1987; Paschal et al., 1987). Finally, the rapid turnover lows fertilization can be traced back to the localization rates, provided by dynamic instability (Mitchison and Kirsch- I of developmental determinants in the egg cytoplasm ner, 1984), allow microtubule arrays to be rapidly changed (Wilson, 1925; Davidson, 1986). The inheritance of a into dramatically new configurations during the transition specific subset of mRNAs and proteins provides the macro- from interphase to mitosis. Taken together, these unique molecular blueprints for the construction of diverse cellular properties of microtubules provide a highly organized and forms with unique functions. In many cases, the compart- yet dynamic network for the anchoring and distribution of mentalization of developmentally significant mRNAs is mRNA to a given region of an egg or embryo. thought to occur through an association with the cytoskele- Recent evidence indicates that specific mRNAs are local- ton. Similarly, in somatic cells, the cytoskeleton provides a ized and transported within oocytes and embryos in a network upon which individual mRNAs can be arranged in microtubule-dependent manner. Microtubules are involved a three-dimensional pattern. The localized synthesis of in the transport of Vgl RNA to the vegetal hemisphere of specific proteins may have important consequences for the stage IV Xenopus oocytes (Yisraeli et al., 1990) and the intrinsic regulation of cell fate and the specification of cellu- perinuclear localization of cyclin B transcripts in synctial lar domains such as axons and dendrites. All the major Drosophila embryos (Raft et al., 1990). The mRNA for the cytoskeletal structures, microtubules, microfilaments, and anterior morphogen, bicoid, is transported from the nurse intermediate filaments, have been implicated in the targeting cells to the anterior margin of the oocyte in a microtubule- and transport of mRNA (reviewed by Jeffery, 1989; Fulton, dependent process (Pokrywka and Stephenson, 1991). Dur- 1993; Singer, 1992; Steward and Banker, 1992; Suprenant, ing the subsequent cellularization of Drosophila embryos, a 1993; Wilhelm and Vale, 1993). network of microtubules sandwiches the RNA coding for the Of the cytoskeletal elements, microtubules are especially segmentation gene product fushi tarazu against the plasma well designed for mRNA localization. Microtubules are membrane, thus acting as a physical barrier for the diffusion asymmetric with a fast-growing (plus) and slow-growing of a developmental determinant (Edgar et al., 1987). Finally, (minus) end. Within the cell, microtubules are generally or- granules containing myelin basic protein mRNA appear to ganized with their minus ends embedded in a microtubule move along microtubule tracts in oligodentrocytic processes organizing center and their plus ends oriented radially to- (Ainger et al., 1993). wards the cell periphery (Euteneuer and Mclntosh, 1981; Specific localization signals in the mRNA itself may be re- Haimo and Telzer, 1981). Along this organized network, quired for mRNA association with the cytoskeleton. For bi- dynein and kinesin generate the forces necessary for micro- coid, Vgl, and nanos (posterior Drosophila morphogen), lo- tubule-based transport towards the minus or plus ends, re- calization signals have been identified in the 3' untranslated spectively (Gibbons and Rowe, 1965; Vale et al., 1985; Lye region of the transcripts (Macdonald and Struhl, 1988; Gott- lieb, 1992; Gravis and Lehman, 1992; Mohry and Melton, Address all correspondence to Dr. Kathy Suprenant, 4010 Haworth Hall, 1992; Macdonald et al., 1993). The 3' untranslated region Department of Physiology and Cell Biology, University of Kansas, of actin mRNA is both necessary and sufficient for the local- Lawrence, KS 66045. Phone: (913) 864-4580; fax: (913) 864-5321. ization of ~-actin to the cell periphery and c~-actin to the © The Rockefeller University Press, 0021-9525/94/11/973/12 $2.00 The Journal of Cell Biology, Volume 127, Number 4, November 1994 973-984 973 perinuclear region of cultured fibroblasts (Kislauski et al., these microtubule preparations, and they can be translated 1993). Each localization signal may have a cytoskeletal into polypeptides in vitro. This reconstituted system should receptor that mediates attachment to and/or motility along prove valuable for dissecting how mRNAs and proteins are a cytoskeletal track. Although such receptors have not been targeted to the cytoskeleton. identified, possible candidates include RNA-binding pro- A brief account of these results has appeared in abstract teins (Schwartz et al., 1992). In Drosophila, three additional form (Suprenant, K. A., and J. Drawbridge. 1991. J. Cell genes, exuperantia, swallow, and staufen, appear to be Biol. 115:341a). necessary for the association of bicoid RNA with the cytoskeleton (Berleth et al., 1988; Stephenson et al., 1988; St. Johnston et al., 1989; Pokrywka and Stephenson, 1991). Materials and Methods The products of these genes may be mRNA chaperones or anchors to the cytoskeleton. Experimental Materials and Solutions Messenger RNA may interact with the cytoskeleton Sea urchins, Strongylocentrotuspurpuratus, were purchased from Marinus, directly, or through other components of the translational Inc. (Long Beach, CA). All nucleotides and analogues were purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN). Ultrapure phenol machinery, such as ribosomes or cytoplasmic ribonucleo- was obtained from Gibco BRL (Gaithersburg, MD). Placental RNasin and protein particles. In many cultured mammalian cells, clus- nuclease-treated, rabbit reticulocyte lysate were purchased from Promega ters of ribosomes surround microtubules, and they are linked (Madison, WI) and Ambion, Inc. (Austin, TX). Primary antibodies were to the microtubule walls by short filaments (Wolosewick and obtained from the following sources: anti-tubulin (DMIA) and anti-actin (C4) from ICN ImmunoBiologicais (Lisle, IL); anti-cytokeratia (BT-571) Porter, 1976; Heuser and Kirschner, 1980; Ris, 1985). In Downloaded from http://rupress.org/jcb/article-pdf/127/4/973/1264027/973.pdf by guest on 27 September 2021 from Biomedical Technologies (Stoughton, MA); anti-kinesin (SUK4) hemipteran insects, ribosomes appear to move along micro- from J. Scholey (University of California, Davis, CA); anti-dynein (71-4.2) tubule tracks from the anterior ovarioles to the developing from D. Asai (Purdue University, Lafayette, IN); anti-ribosome (40S) and oocytes (reviewed in Stebbings, 1986). In the mitotic ap- anti-PABPs from M. Winkler (University of Texas, Austin, TX). These an- paratus, ribosome-like particles are attached to adjacent mi- tibodies were used at the following dilutions: DM1A (1:1000); C4 (1:250); BT-571 (1:200); SUK4 (1:500); 71-4.2 (1:500); anti-ribosome (1:500); and crotubules by fine filamentous arms (Goldman and Rebhun, anti-PABP (1:500). Secondary antibodies were purchased from Zymed 1969; Salmon and Segall, 1980; Silver et al., 1980; Laboratories, Inc. (South San Francisco, CA). All other reagents were ob- Hirokawa et al., 1985; Suprenant et al., 1989). tained from the Sigma Chemical Co. (St. Louis, MO) and Research Or- We have developed a novel model system to study how and ganics (Rochester, NY). RNase-free glassware and plasticware were used why components of the translational machinery interact with throughout this study. microtubules. Unfertilized sea urchin eggs are metabolically quiescent cells that contain large stores of unassembled Gametes and Embryo Culture microtubule protein as well as maternal mRNAs, of which Eggs and sperm were obtained by intracoelomic injection of 0.55 M KC1. only 1-5 % are actively being translated. The ionic events at Sperm were collected "dry" and stored on ice until needed. Eggs were shed into MiUipore-filtered Instant Ocean (MF-IO) artificial sea water (Aquar- fertilization activate protein synthesis (reviewed by Rosen- ium Systems, Mentor, OH), and were harvested by gentle centrifugation thai and Wilt, 1987), and they promote the polymerization (100 g, 2 min). After three washes in artificial sea water, egg jelly coats were of microtubules (reviewed by Schatten, 1984). Both pro- removed in "19:1" (500 mM NaCI, 27 mM KC1, 2 mM EDTA, pH 7.8). cesses appear to be driven by the alkalinization of the

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