InteractiveFly: GeneBrief

ensconsin: Biological Overview | References

BIOLOGICAL OVERVIEW

Ensconsin is a conserved microtubule-associated protein (MAP) that interacts dynamically with microtubules, but its cellular function has remained elusive. This study shows that Drosophila ensconsin is required for all known kinesin-1-dependent processes in the polarized oocyte without detectable effects on microtubules. ensconsin is also required in neurons. Using a single molecule assay for kinesin-1 motility in Drosophila ovary extract, it was shown that recruitment to microtubules and subsequent motility is severely impaired without ensconsin. Ensconsin protein is enriched at the oocyte anterior and apically in polarized epithelial cells, although required for localization of posterior determinants. Par-1 is required for ensconsin localization and directly phosphorylates it at conserved sites. These results reveal an unexpected function of a MAP, promoting productive recruitment of a specific motor to microtubules, and an additional level of kinesin regulation. Furthermore, spatial control of motor recruitment can provide additional regulatory control in Par-1 and microtubule-dependent cell polarity (Sung, 2008).

Asymmetric cell polarization is fundamental to embryonic development and to the functions of epithelial cells. For cells that are large, like some oocytes, or have long extensions, such as neurons, the microtubule cytoskeleton contributes to polarity and provides the means for directional long-distance transport. As microtubules are inherently polarized, spatial control of their nucleation and growth can give a polarized cytoskeleton. This polarity is then 'read out' by minus-end-directed motors, primarily the dynein motor complex, and plus-end-directed motors, such as classical kinesins (Sung, 2008).

Microtubules interact with, and are regulated by, a large number of proteins. Apart from motors, diverse families of conserved microtubule-associated proteins (MAPs) have been identified. Their functions can vary, affecting microtubule growth, stability, or interactions. They may also affect the directional motors - for example, acting as 'roadblocks.' Tau, a well-studied MAP involved in neurodegeneration, interferes with kinesin-1 transport when overexpressed in cells (Ebneth, 1998; Stamer, 2002), and with the processive movement of purified kinesin-1 motors on microtubules (Seitz, 2002; Vershinin, 2007). This shows that Tau can block the kinesin-1 motor, but whether this is its physiological function remains a matter of debate. One complication in assigning biological function to MAPs lies in the difficulty of bridging the gap between knowledge obtained from detailed in vitro experiments that use artificially naked microtubules and incomplete motor complexes and the physiological effects that can be observed in cells and tissues (Sung, 2008).

Mammalian Ensconsin, also called E-MAP-115 for epithelial microtubule-associated protein of 115 kD, was isolated biochemically by virtue of its ability to tightly associate with microtubules (Masson, 1993; Bulinski, 1994). This study used the name ensconsin to avoid confusion with the unrelated EMAP (echinoderm MAP). Ensconsin binds along the length of microtubules, and early studies suggested that it might stabilize microtubules. However, this idea has not been supported by subsequent experiments involving less extreme overexpression (Faire, 1999). In addition, live imaging showed that the association between ensconsin and microtubules is very dynamic in cells and appears to be regulated by phosphorylation events (Faire, 1999; Bulinski, 2001). Phosphorylation of ensconsin and its microtubule association is regulated during the cell cycle (Masson, 1995). Mice mutant for ensconsin/E-MAP-115 are viable, but have defects in microtubule-rich structures in spermatogenesis (Komada, 2000). More widespread functions might be obscured by genetic redundancy, however, as mammalian genomes encode a related protein, RPRC1. Drosophila has only one gene of this family, which is investigated here (Sung, 2008).

The oocyte of Drosophila is a well-studied example of microtubule-dependent cell polarization. At mid-to-late oogenesis, localization of the anterior determinant bicoid requires the anterior microtubules and the dynein motor complex, whereas posterior determinants Staufen and oskar mRNA require kinesin-1, a plus-end-directed motor. Recent analysis indicates that oskar mRNA transport is primarily mediated by kinesin-1, itself performing a random biased walk along a weakly polarized cytoskeleton (Zimyanin, 2008). In many contexts, the Par proteins are important for polarity, establishing mutually exclusive cortical domains, and affecting the cytoskeleton. Setting up oocyte polarity also requires Par-1 kinase, which itself is enriched at the posterior. Par-1 activity, together with the phosphorylation-induced binding of 14-3-3/Par-5 to target proteins, restricts proteins, such as Bazooka/Par-3, to the apical domain in epithelial cells. A similar mechanism may act downstream of Par-1 in the germline (Benton, 2003), but here the relevant Par-1 targets are not known. A link from Par-1 to microtubules appears conserved even if not fully understood. A mammalian homolog of Par-1, microtubule affinity regulating kinase (MARK), was identified based on its ability to phosphorylate microtubule-associated proteins (Drewes, 1997) and thereby affect microtubules. The MAP Tau is phosphorylated by Par-1/MARK in both mammalian and Drosophila cells (Drewes, 1997; Doerflinger, 2003). However, Tau is not required for development or for Drosophila oogenesis (Doerflinger, 2003). Par-1/MARK may have multiple targets that impinge on the microtubule cytoskeleton. This study found that ensconsin/E-MAP-115 is a physiologically significant Par-1 target in the oocyte. Par-1 regulates ensconsin localization, which, in turn, is required for effective kinesin-1-dependent transport (Sung, 2008).

In a screen for genes affecting development of the Drosophila female germline, a PiggyBac insertion was identified in the gene CG14998. The encoded protein is similar to mouse ensconsin/E-MAP-115, with two regions of higher similarity that are called ensconsin homology region (EHR)-1 and EHR2. For both mammalian and Drosophila proteins, multiple isoforms exist due to alternate splicing immediately before and after EHR1. With a microtubule pelleting assay routinely used to identify MAPs, the endogenous CG14998 protein was found to associate with microtubules in embryo extracts as well as in ovary extract. In mouse ensconsin, the N-terminal 200 amino acids were responsible for microtubule association (Masson, 1993). Similarly, in CG14998, a truncated protein removing EHR2 but retaining EHR1 (Ens-X) still associated with microtubules when expressed in the ovary, whereas removing EHR1 but retaining EHR2 (Ens-C) abrogated microtubule binding, whether the protein was expressed at modest or high levels. Finding CG14998 to be a likely functional ortholog of mouse ensconsin/E-MAP-115, it was named ensconsin (Sung, 2008).

The microtubule cytoskeleton is complex and dynamic, with extensive regulation of microtubule growth and turnover. It is not clear, however, whether the resulting polarized microtubules are simply roads to be freely used by motors or whether there is significant crowding on them and assistance is needed to get on. In this study, this study provides evidence that a MAP may function to stimulate productive interaction of a specific directional motor, kinesin-1, with microtubules in vivo. This was demonstrated using a combination of genetic analysis and single-molecule imaging performed in cell extracts. That Ensconsin is required for efficient kinesin-1-dependent transport in vivo, irrespective of which cargo is being transported, was demonstrated by the specificity of the mutant phenotypes. This was shown in detail for the oocyte, but analysis of neuronal phenotypes suggests that the function of Ensconsin is general. The kinesin motility assay in extract allowed direct analysis of physiologically active kinesin with and without Ensconsin present under conditions close to the cellular environment, but on experimentally defined microtubule tracts. Previous studies have shown that some MAPs can regulate the dynamics of microtubules themselves (Mandelkow, 1995), and that others can act as 'roadblocks,' inhibiting motor traffic (Ebneth, 1998; Stamer, 2002; Seitz, 2002; Vershinin, 2007). Aiding microtubule motor activity somewhat unexpected function for a MAP, and has not been shown previously (Sung, 2008).

The difference in the effect of Ensconsin on full-length kinesin versus the short 'constitutively active' form highlights the importance of finding appropriate ways to analyze complete and functional motor proteins in order to understand their regulation. The method described here, using functional kinesin and cell extracts, should be helpful in this regard. Importantly, the results with and without Ensconsin reveal an additional level of regulation of the inherently very active motor, kinesin-1. Binding of specific cargo is thought to stimulate motor activity by relieving autoinhibition in classical kinesins. This may ensure that only occupied kinesin motors interact with microtubules. A dedicated regulator, such as Ensconsin, can provide spatial and temporal control of the motor, regardless of the cargo. Ensconsin protein accumulation is itself spatially controlled, and microtubule association of Ensconsin may be controlled by phosphorylation (Masson, 1995; Faire, 1999; Bulinski, 2001), allowing recruitment of kinesin/cargo complexes to microtubules to be intricately regulated. As the truncated kinesin is active regardless of ensconsin, both in vivo and in extracts, this additional level of control also appears to operate as relief of inhibition. This implies that most kinesin in the cell is in an inactive state, and multiple positive inputs converge to allow actual motility (Sung, 2008).

Tau is a MAP that, like Ensconsin, binds along microtubules, and, in some assays, is able to stabilize microtubules. Tau has been extensively studied due to its association with neurodegeneration, but its actual biological function is still somewhat unclear. In cells, moderately overexpressed Tau inhibits the function of classical kinesin. Tau may inhibit the productive attachment of kinesin-1 motor to microtubules and thereby also processivity of movement for cargoes with more that one kinesin motor. Ensconsin does the opposite. Such potentially counteracting MAPs may allow fine-tuned control over trafficking along microtubules, which would be of particular importance in neurons and other large, polarized cells (Sung, 2008).

The present findings for Drosophila Ensconsin are likely to apply to mammalian ensconsin as well. Mammalian ensconsin was found to associate dynamically with microtubules along their length, and modest overexpression did not affect microtubule stability (Faire, 1999; Bulinski, 2001). Similarly, this study did not observe overt defects in microtubule density upon removal of ensconsin in vivo. Like the fly protein, mammalian ensconsin is apically localized in a polarized epithelium (Vanier, 2003) and contains potential 14-3-3 binding sites between EHR1 and EHR2, which are likely Par-1 phosphorylation sites. No clear requirement was found for Ensconsin in the follicular epithelium, but it may be subtle or redundant with other regulators. In both mammals and flies, Par-1/MARK is unlikely to be the only kinase regulating ensconsin. This is indicated by the heavy cell cycle-dependent phosphorylation of mammalian ensconsin affecting its interaction with microtubules (Masson, 1995), as well as the 'background' (Par-1-independent) phosphorylation seen in the current kinase assays with oocyte proteins. Finally, EHR1 is responsible for microtubule binding in both mammalian and Drosophila ensconsin. The second conserved domain, EHR2, may be responsible for the specific effect on kinesin. A detailed biophysical analysis will be needed to determine exactly how Ensconsin stimulates both recruitment and subsequent motility of full-length kinesin. A simple hypothesis would be recruitment by direct physical interaction, which has so far not been detected. However, given that ensconsin also has a positive effect on kinesin movement, their interaction would most likely be transient and/or regulated -- and possibly not straightforward to observe (Sung, 2008).

Localization of ensconsin is controlled at multiple levels, including mRNA localization, Par-1 phosphorylation, and a dependency on microtubules. The Ens-mut mislocalization shows that at least some of the effects of Par-1 on ensconsin are direct. The clear microtubule dependency in the oocyte indicates that Par-1 phosphorylation is not sufficient for biased Ens localization; it requires transport or other microtubule-dependent events as well. Spatially, Par-1 negatively affects Ensconsin, and this agrees with the phenotype of mislocalized Ensconsin resembling that of Par-1 loss of function. Why is Ensconsin subject to this regulation, and how may this contribute to polarity? A recent study argues convincingly that the posterior determinant oskar is localized primarily as a cargo of kinesin-1, with motor-cargo particles moving inefficiently by biased random walk toward the posterior (Zimyanin, 2008). The implication is that microtubules in the oocyte have a net polarity in the expected direction (more minus ends anteriorly), but it is only a weak bias with many microtubules pointing in other directions. A higher level of Ensconsin at the anterior/lateral cortex can promote more productive interactions of kinesin-cargo complexes with microtubules in this region, and, hence, contribute to the bias of posteriorly directed events. At the posterior, microtubule density is relatively low, but kinesin-1 accumulates over time. Low levels of Ensconsin (thus, very little active kinesin-1) will discourage active 'backwards' transport of kinesin cargoes. The asymmetric Ensconsin distribution can thus contribute to asymmetry in microtubule-dependent transport (Sung, 2008).

The oocyte is a large and dynamically polarized structure likely utilizing multiple overlapping mechanisms of polarity establishment and maintenance. Microtubule-dependent asymmetry and polarity can be affected by microtubule density, as well as by bias in orientation. Both processes may be modulated by Par-1. The function and regulation of Ensconsin described in this study indicate that regulators of microtubule-dependent polarity need not affect microtubules themselves, but may also control the effectiveness of the directional motors that 'read out' the polarity of the microtubules (Sung, 2008).

Ensconsin/Map7 promotes microtubule growth and centrosome separation in Drosophila neural stem cells

The mitotic spindle is crucial to achieve segregation of sister chromatids. To identify new mitotic spindle assembly regulators, this study isolated 855 microtubule-associated proteins (MAPs) from Drosophila melanogaster mitotic or interphasic embryos. Using RNAi, 96 poorly characterized genes were screened in the Drosophila central nervous system to establish their possible role during spindle assembly. Ensconsin/MAP7 mutant neuroblasts were found to display shorter metaphase spindles, a defect caused by a reduced microtubule polymerization rate and enhanced by centrosome ablation. In agreement with a direct effect in regulating spindle length, Ensconsin overexpression triggered an increase in spindle length in S2 cells, whereas purified Ensconsin stimulated microtubule polymerization in vitro. Interestingly, ensc-null mutant flies also display defective centrosome separation and positioning during interphase, a phenotype also detected in kinesin-1 mutants. Collectively, these results suggest that Ensconsin cooperates with its binding partner Kinesin-1 during interphase to trigger centrosome separation. In addition, Ensconsin promotes microtubule polymerization during mitosis to control spindle length independent of Kinesin-1 (Gallaud, 2014).

Search PubMed for articles about Drosophila Ensconsin

Benton, R., Palacios, I. M. and St. Johnston, D. (2002). Drosophila 14-3-3/PAR-5 is an essential mediator of PAR-1 function in axis formation. Dev. Cell 3: 659-671. PubMed ID: 12431373

Bulinski, J. C. and Bossler, A. (1994). Purification and characterization of ensconsin, a novel microtubule stabilizing protein. J. Cell Sci. 107: 2839-2849. PubMed ID: 7876351

Doerflinger, H., et al. (2003). The role of Par-1 in regulating the polarised microtubule cytoskeleton in the Drosophila follicular epithelium. Development 130: 3965-3975. PubMed ID: 12874119

Drewes, G., et al. (1997). MARK, a novel family of protein kinases that phosphorylate microtubule-associated proteins and trigger microtubule disruption. Cell 89: 297-308. PubMed ID: 9108484

Ebneth, A., et al. (2008). Overexpression of Tau protein inhibits kinesin-dependent trafficking of vesicles, mitochondria, and endoplasmic reticulum: implications for Alzheimer's disease. J. Cell Biol. 143: 777-794. PubMed ID: 9813097

Faire, K., et al. (1999). E-MAP-115 (ensconsin) associates dynamically with microtubules in vivo and is not a physiological modulator of microtubule dynamics. J. Cell Sci. 112: 4243-4255. PubMed ID: 10564643

Gallaud, E., Caous, R., Pascal, A., Bazile, F., Gagne, J. P., Huet, S., Poirier, G. G., Chretien, D., Richard-Parpaillon, L. and Giet, R. (2014). Ensconsin/Map7 promotes microtubule growth and centrosome separation in Drosophila neural stem cells. J Cell Biol 204: 1111-1121. PubMed ID: 24687279

Komada, M., et al. (2000). E-MAP-115, encoding a microtubule-associated protein, is a retinoic acid-inducible gene required for spermatogenesis. Genes Dev. 14: 1332-1342. PubMed ID: 10837026

Mandelkow, E. and Mandelkow, E. M. (1995). Microtubules and microtubule-associated proteins. Curr. Opin. Cell Biol. 7: 72-81. PubMed ID: 7755992

Masson, D. and Kreis, T. E. (1993). Identification and molecular characterization of E-MAP-115, a novel microtubule-associated protein predominantly expressed in epithelial cells. J. Cell Biol. 123: 357-371. PubMed ID: 8408219

Masson, D. and Kreis, T. E. (1995). Binding of E-MAP-115 to microtubules is regulated by cell cycle-dependent phosphorylation. J. Cell Biol. 131: 1015-1024. PubMed ID: 7490279

Seitz, A., (2002). Single-molecule investigation of the interference between kinesin, Tau and MAP2c. EMBO J. 21: 4896-4905. PubMed ID: 12234929

Stamer, K. et al. (2002). Tau blocks traffic of organelles, neurofilaments, and APP vesicles in neurons and enhances oxidative stress. J. Cell Biol. 156: 1051-1063. PubMed ID: 11901170

Sung, H. H., et al. (2008). Drosophila ensconsin promotes productive recruitment of Kinesin-1 to microtubules. Dev. Cell. 15(6): 866-76. PubMed ID: 19081075

Vanier, M. T. et al. (2003). Expression and distribution of distinct variants of E-MAP-115 during proliferation and differentiation of human intestinal epithelial cells. Cell Motil. Cytoskeleton 55: 221-231. PubMed ID: 12845596

Vershinin, M., et al. (2007). Multiple-motor based transport and its regulation by Tau. Proc. Natl. Acad. Sci. 104: 87-92. PubMed ID: 17190808

Zimyanin, V. L. et al. (2008). In vivo imaging of oskar mRNA transport reveals the mechanism of posterior localization. Cell 134: 843-853. PubMed ID: 18775316

date revised: 22 January 2022

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