The cytoskeleton in mRNA localization and cell differentiation

396

The cytoskeleton in mRNA localization and cell differentiation Kim Nasmyth* and Ralf-Peter Jansent Asymmetric

distribution of cytoplasmic

messenger

RNAs has been implicated

of cell differentiation. direct mRNA

Microtubules

proteins and in several instances

have been suggested

to

localization in Drosophila and Xenopus oocytes

but motor proteins that might transport mRNAs

have not

yet been identified. Recent data imply that in Drosophila,

Caenorhabditis elegans and budding yeast, proteins of the actin cytoskeleton,

including unconventional

active roles in the segregation

myosins, play

of differentiation

factors and

mRNAs.

mRNAs and determinants [4**-7D*,8,9]. It was thought that microtubules are generally required for long-distance transport processes whereas microfilaments are involved in anchoring of RNAs and other factors. However, new data also emphasize the role of microfilaments and actin-interacting proteins in mRNA transport and protein localization. This review will discuss the current evidence for the role of either microtubule-interacting or actin-interacting proteins in localizing determinants and mRNAs encoding determinants.

Microtubules Addresses Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria ‘e-mail: [email protected] te-mail: [email protected] Current Opinion in Cell Biology 1997, 9:396-400 http://biomednet.com/elecref/0955067400900396 0 Current Biology Ltd ISSN 0955-0674 Abbreviations cytoplasmic tropomyosin II cTmll NMY-2 nonmuscle myosin II partitioning defective w Xenopus kinesin-like protein 1 Xklpl

Introduction Asymmetric cell divisions contribute to the generation of different cell types during the development of multicellular organisms. Differences in daughter cells that arise from asymmetric divisions can be generated by an asymmetric localization of the division plane, thus producing daughter cells of different sizes. Alternatively, differences can arise due to asymmetric distribution of cell fate determining factors prior to cell division [1,2]. In organisms like Drosophila or Xenopw, the mRNAs encoding the determinants, rather than proteins themselves, are often asymmetrically distributed. Segregating mRNAs instead of proteins has the advantage of localizing protein synthesis to the site of the protein’s function and may allow the local assembly of the synthesized protein into macromolecular complexes. Localized protein synthesis is in fact not unique to cells of the developing embryo, but is also found in somatic cells where it plays a role in protein sorting [3]. How are localized mRNAs and proteins transported to their destination site? There are three cytoskeletal filament filaments, systems in the cell, namely, intermediate microfilaments and microtubules. Studies in Caenorkabd&is elegans, DrosopMa melanogaster and Xenopus laevis embryos, as well as in fibroblasts and neurons, have implicated microtubules and microfilaments in localizing

Microtubule-dependent

mRNA localization in Dmsophi/8

Several lines of evidence suggest that microtubules play an important role during localization of maternal mRNAs in Drosophila [lo]. Rearrangement of the microtubular network correlates with the localization of maternal mRNAs. Among other mRNAs, bicoid, gut-ken, Bicaudal-D and oskar mRNAs are transported early during the oocyte’s development from the nurse cells to the oocyte. Later in oocyte development, bicoid and Bicaudal-D move from the posterior to the anterior end of the oocyte. These two movements correlate with the orientation of the microtubule network and occur from the plus to the minus end of microtubules. Early in oocyte development, the microtubule nucleation site (the minus end) is located at the posterior of the oocyte with the microtubules pointing towards the nurse cells [4”,10,11]. Later, the orientation of microtubules undergoes a rearrangement with the nucleation site being found at the anterior of the oocyte. In both cases, mRNAs move towards the minus end of microtubules. A second piece of evidence for the involvement of microtubules in mRNA localization comes from experiments in which female flies were treated with inhibitors of microtubule polymerization. Such treatment leads to mislocalization of several maternal mRNAs (see [4**,.5”] and references therein). However, drug treatment is rather drastic and is hard to interpret because the observed effects of microtubule depolymerization could be rather indirect by simply upsetting the oocyte’s cytoskeleton. Transport of mRNAs along microtubules requires microtubule-dependent motor proteins. One such motor protein is cytoplasmic dynein, which moves towards microtubule minus ends. The transport rates of KIO, bicoid and oskar mRNAs are consistent with dynein being the microtubule-dependent motor protein that transports these mRNAs from the nurse cells to the oocyte and that is required for correct mRNA localization within the oocyte itself [12]. A candidate dynein could be the dynein heavy chain protein DHC64C which localizes to the early oocyte when maternal mRNAs are transported from the nurse

The cytoskeleton in mRNA localization and cell differentiation Nasmyth and Jansen

cells into the oocyte [13]. Other experiments indicate that kinesin could be involved in RNA transport [14]. Although these data are suggestive, we still lack really hard evidence that mRNAs are actually transported by any of the above-mentioned microtubule-dependent motor proteins.

Microtubule-dependent mRNA localization in vertebrates Microtubule-dependent RNA localization is also found in vertebrates. Xenopus /aevis oocytes contain more than 10 different RNAs that are localized either to the vegetal or to the animal pole. The transport of Vgl mRNA, which codes for a member of the transforming growth factor-p (TGF-P) family, is abolished by treating oocytes with microtubule-depolymerizing drugs such as nocodazole and colchicine [15]. The microtubule-dependent transport process is possibly conserved between frogs and mammals, as tau mRNA from mammalian neurons, when injected into oocytes, can take the same microtubule-dependent route to the vegetal cortex as does VgZ mRNA itself [16’]. As in DrosopMa, we know of no microtubule-dependent motor that could transport VgZmRNA. However, a specific Vgl RNA binding protein of 69 kDa has been identified that mediates Vgl mRNA association with microtubules and itself has microtubule-binding activity [17*]. A protein with similar characteristics, the mouse RNA-binding protein TB-RBP (testis/brain RNA-binding protein), might serve as an adaptor protein for the association with microtubules of certain mRNAs in brain and testis [18]. However, the molecular nature and especially the exact function of the Vgl RNA binding protein and TB-RBP in mRNA localization are still unknown. In addition to their putative role in mRNA localization in Xenopus, microtubules are required for the aggregation of germ plasm in the oocyte. Germ plasm is a specialized part of the cytoplasm that will eventually give rise to the germ cell line. It consists of several known RNAs, fibrillar material and mitochondria. Germ plasm is found as isolated clusters at the vegetal pole that aggregate during the first embryonic cleavages to form a large complex. Aggregation of germ plasm clusters requires the function of a kinesin-like microtubule-dependent motor protein, Xklpl (Xenopus kinesin-like protein 1) [19**]. Xklpl is probably directly involved in the transport of the individual clusters towards each other as it colocalizes with the germ plasm at the time of aggregation and its depletion blocks germ plasm aggregation without disrupting the overall structure of the microtubule network. With the exception of Xklpl, we currently do not know the key components of microtubule-dependent RNA transport, that is, the motor proteins. Genetic screens in Drosopkh’a and affinity-purification approaches using localized mRNAs as a ‘bait’ have not yet come up with microtubule motor proteins that_like Xklpl - are not required for general functions’~of the microtubule

cytoskeleton but have the specific mRNAs or determinants.

function

397

of moving

Actin Do actin filaments also play a role in mRNA segregation? A large fraction of poly(A)+ RNA and polyribosomes are associated with actin microfilaments [ZO] but these polyribosomes and poly(A)+ RNA are usually not localized to a particular site within the cell. One exception occurs during early embryogenesis of the algae Fzms, where the bulk of the poly(A)+ RNA is unequally distributed upon division. The asymmetric distribution of bulk mRNA is abolished by treatment with the microfilament-disrupting drug cytochalasin D [Zl], indicating the importance of actin in this process. In Xenopus laevis oocytes, the results of actin filament disruption with cytochalasin D suggested that the peripheral actin cytoskeleton is important for the anchoring of a set of individual mRNAs to the oocyte’s cortex (see Fig. 1) but not for the transport to the cortex [15]. Genetic studies in Drosopkila have also implicated microfilaments in oocyte mRNA and protein localization. Mutations in the cytoplasmic tropomyosin II (cTmI1) disrupt both germ cell determination (at the oocyte’s posterior end) and head formation at the anterior [22”,23”]. cTmI1 mutants can no longer localize oskar mRNA and Staufen protein (an mRNA-binding protein) to the oocyte’s posterior end (see Fig. 1) and consequently have mislocalized Oskar protein which normally serves as a landmark for the establishment of the anterior-posterior axis and for germ cell formation. It has been suggested that tropomyosin is not involved in the actual transport of the mRNA but is instead required to sequester oskar mRNA to the actin-rich posterior cortex of the oocyte. However, a direct role of cTmI1 in oskar mRNA localization is still possible. Profilin, another actin-binding protein, might also be involved in localizing oskar mRNA to the posterior pole as mutants in dickadee (the gene encoding Drosophila profilin) disrupt the posterior localization of oskar mRNA [24]. In addition to mislocalizing o.skar mRNA, profilin mutants cause premature ooplasmic streaming. Ooplasmic streaming is thought to mix the oocyte’s content and to facilitate diffusion processes. It has been suggested that ooplasmic streaming is involved in mRNA localization [5**]. However, if ooplasmic streaming occurs too early (as in profilin mutants) it might in fact interfere with proper mRNA localization. Mutations in cappucino result in a very similar phenotype to that of profilin mutations [25]. It is not clear at the moment if oskar mRNA mislocalization in profilin and cappucino mutants is a result of the effect of profilin and cappucino on the timing of ooplasmic streaming or is due to a more direct role of these proteins in mRNA transport.

398

Nucleus and gene expression

Fiaure 1

Xenopus

,

Asymmetric localization dependent on

oocyte

0 0

Drosophila

Vg7 mRNA actin microfilaments (only for anchoring at vegetal cortex)

oocyte oskar mRNA Staufen

cytoplasmic tropomyosin II, profilin

by disruption of microfilaments or microtubules. However, the orientation of the crescent relative to the division plane is altered by microfilament disruption. The same effect is seen in mutants defective in inscuteable [28**]. Inscuteable protein localizes in an actin-dependent manner to the cell cortex and is required to coordinate the orientation of the mitotic spindle, the division plane, and the Numb/Prosper0 crescent. Actin filaments could therefore control, via Inscuteable localization, the asymmetric division of Dmsop/ri/a neuroblasts. It is interesting that microfilament disruption in C. elegans zygotes also changes the early asymmetric cleavage pattern to a symmetric pattern [29].

Actin-dependent C. elegans

zygote

0

PAR-l PAR-2

nonmuscle myosin NMY-2

D PAR-3 S. cerevisiae Myo4p, Bnil p, She4p, tropomyosin, profilin, actin 0 1997 Current Odnion in Cell Bidw

Examples of actin-dependent asymmetric distribution both of protein determinants and of mRNAs that encode determinants. Black areas indicate the localization of particular proteins and mRNAs. The results of drug treatment experiments and genetic screens suggest an important role of the actin cytoskeleton in determinant segregation. The identified proteins that are known to be needed for localization of particular determinants/mRNAs are shown at the right of the figure. Disruption of actin microfilaments by cytochalasin D treatment leads to the release of Vg7 mRNA from the vegetal cortex of Xenopus laevis oocytes, indicating that actin is required for the cortical anchoring of this mRNA (transport itself of Vg7 mRNA in Xenopus is microtubule-dependent [151). Exposure of C. elegans embryos to cytochalasin D causes improper distribution of RNA-containing P granules that are necessary for germ cell formation (not shown here) [29]. Genetic studies in Drosophila, C. elegans and S. cerevisiae demonstrate that mutants of actin cytoskeletal proteins (including two unconventional myosins and cytoplasmic tropomyosins) inhibit the asymmetric distribution of osksr mRNA and Staufen protein (in Drosophila [22**,23*‘,24]), PAR-l, PAR-2 and PAR-3 proteins (in C. elegans [33”]), and Ash1 protein and mRNA (in S. cerevisiae [35**,36**,37]; R Long, personal communication).

In Drosop/ri/a, microfilaments are also involved in controlling the orientation of division planes in somatic cells and, in doing so, in asymmetric segregation of the determinants Numb and Prosper0 [26,27,28”]. During asymmetric divisions of neuroblasts and peripheral sensory organ precursor cells, Numb and Prosper0 proteins, which determine the correct cell fate of neurons, localize asymmetrically as a crescent at the cortex of the dividing cell. As the division plane is parallel to this crescent, the two proteins are inherited by only one daughter cell [26,27]. The asymmetric accumulation of both proteins is unaffected

motor proteins

In contrast to the lack of microtubule-dependent motors, two unconventional myosins, or actin-dependent motor proteins, have recently been characterized that are required to localize determinants and to generate cells with different fates. In 15’. e/egans, the products of the par (partitioning defective) genes are required for the asymmetric cleavage pattern of the early embryo and are themselves asymmetrically distributed during early embryogenesis [30]. In the zygote, par-l, which encodes a serine/threonine kinase, is enriched at the posterior cortex [31]. It has not been proven if the asymmetric distribution of PAR-l protein results from protein segregation or is a consequence of asymmetric par-2 mRNA distribution as the latter has not yet been localized by in situ hybridization. Asymmetric PAR-l protein distribution requires the function of other PAR proteins, PAR-Z and PAR-3 [32]. In addition, an unconventional myosin of the subclass II [33”] is essential for proper PAR-1 localization (see Fig. 1). Nonmuscle myosin II (NMY-2) was identified on the basis of its in vitro binding to the conserved PAR-l carboxy-terminal domain. nmy-2 mRNA depletion abolished the posterior localization of PAR-l protein. NMY-2 is presumably an actin-dependent motor protein and could have a direct role in localizing the determinant PAR-l to the posterior half of the zygote. However, PAR-l localization is dependent on PAR-Z and PAR-3 (but not vice veersa) and disruption of the nmy-2 mRNA also disrupts the localization pattern of PAR-2 and PAR-3. Therefore, one could think of additional roles of NMY-2. It could localize PAR-l, PAR-2 and PAR-3 proteins independently of each other or it could localize PAR-2 and PAR-3 which in turn localize PAR-l. It will be interesting to see if NMY-2 can also directly associate with other PAR proteins. The budding yeast protein Myo4, a type V unconventional is essential for the asymmetric distribution myosin, of the transcriptional regulator Ashlp [34’,3.5”,36”]. Ashlp preferentially accumulates in only one of the two nuclei following anaphase. In this nucleus, Ashlp inhibits expression of the HO gene (which encodes an endonuclease that initiates mating-type switching). This

The cytoskeleton

in mRNA

asymmetric distribution depends on five nonessential SHE genes, at least three of which encode novel cytoskeletal proteins (see Fig. 1). One of them is Myo4p, an unconventional myosin that accumulates in the cytoplasm of the daughter cell. Another She protein, SheS/Bnilp, is required for several aspects of microfilament function in yeast and interacts with Rholp, a GTP-binding protein that is important for the polarization of the actin cytoskeleton [37]. Like SheSp/Bnilp, She4p is also involved in actin-dependent processes in yeast as s/re4 mutants show impaired cytokinesis and endocytosis, both actin-dependent processes in budding yeast [35”,38]. Furthermore, other actin-interacting proteins, including tropomyosin and profilin, in addition to actin itself are also required for the proper distribution of Ashlp (RP Jansen, unpublished data). In contrast to microfilaments, astral microtubules are not involved in this process. Is Ash1 protein transported by the She proteins? As Ashlp is a nuclear transcription factor and the She proteins are cytoskeletal proteins, this seems unlikely. Recent experiments suggest that the localization of ASH1 mRNA might account for the asymmetric distribution of the protein. ASH2 mRNA is localized in a Myo4p-dependent manner to a small arc at the cortex of the daughter cell (R Long, R Singer, persona1 communication). The implication is that the unconventional My04 myosin might directly localize ASH2 mRNA to the daughter cell cortex. However, it has not been rigorously demonstrated so far that the ASH2 mRNA itself is transported by Myo4p.

localization

and future directions

The past two years have seen important progress in the analysis of &acting elements within mRNAs that are required for mRNA localization [4”-7”]. In addition, it is clear now that mRNA and determinant localization requires cytoskeletal filaments. It is no surprise that both microfilaments and microtubules are involved in these processes as both cytoskeletal systems can cooperate during transport [39]. Initial experiments using drug treatment to disrupt microfilaments or microtubules implicate microtubules as the major filament system for localization. However, drug treatment often has pleiotropic effects, making the interpretation of data from such experiments difficult. In contrast, genetic screens can identify factors that are specifically required for the localization of individual mRNAs and determinants. Recent genetic screens in yeast and DrosopMa have identified proteins of the actin cytoskeleton that are required for the localization of both mRNAs and determinants. What will be the main goals of the field for the future? Clearly, an important one will be the characterization of proteins that are involved in anchoring mRNAs to the cytoskeleton and in binding mRNAs to motor proteins during transport. The most challenging goal, however, will be the identification of motor proteins that move mRNAs or other determinants along the cytoskeleton.

Nasmyth and Jansen

399

So far, only three candidate motor proteins have been identified, namely, Xenopus Xklpl, C. elegans NMY-2 and budding yeast Myo4, two of them being actin-dependent motors. However, none of them has been shown to directly participate in mRNA or determination-factor localization. However, it is a promising start as both C. elegons and yeast represent model systems with powerful genetics that will facilitate the characterization of the known motors and the isolation of interacting partners. Identification of ‘RNA transporters’ in Drosopk’a with its well characterized RNA-localization system is eagerly awaited.

Acknowledgements We want to thank U van Ahsen and I Gonzalez for critically reading the manuscript. We would like to apologize to all colleagues in the field whose work has not been discussed due to limited space. K Nasmyth is supported by the Austrian Industrial Research Promotion Fonds.

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