Presentation on theme: "Bidirectional cargo transport: assessing molecular motor coupling and obstacle encounter using a novel in vitro assay for microtubule-based mRNP transport."— Presentation transcript:
Bidirectional cargo transport: assessing molecular motor coupling and obstacle encounter using a novel in vitro assay for microtubule-based mRNP transport Harish Chandra Soundararajan* and Simon L Bullock MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH 1Introduction2Characterizing the dynamic properties of localizing mRNA using RAT-TRAP Asymmetric localization of a subset of proteins within the cytoplasm is achieved by cytoskeletal transport of their mRNA transcripts (1). Studying transport mechanisms in vivo is challenging because individual microtubules (MTs), cytoskeletal motor components and small mRNA:protein complexes (mRNPs) cannot be visualized readily. Here, building on an in vitro transport assay previously developed in our lab (2), I describe a novel technique to study transport of bidirectional mRNPs assembled in Drosophila embryo extracts termed mRNA Transport by Tethered RNA Purification (RAT-TRAP). Using the assay we are addressing the following questions: how do mRNPs navigate a complex cytoskeletal environment (3)? Do reversals of cargo represent tug-of-war or coordinated switching between opposite polarity motors (4)? To this end we use static motors (rigor kinesin mutant) as obstacles to asasay its effects on the dynamics of an endogenously localizing mRNA (5). Further, to investigate the mechanism of reversals we perform correlation analysis on the minus-end and plus-end dynamics of these mRNPs. Run lengths were significantly greater in the minus-end than the plus-end direction (Mann Whitney test p<0.001) Plus-end velocity was significantly greater than minus-end velocity (Mann Whitney test p<0.001) In vivo dynamic properties of mRNPs have been recapitulated in vitro hairy mRNPs have a population of puncta that are extremely long (as long as 26μm) and unidirectional 3Dynamic properties of localizing mRNA with rigor kinesin (kinesin-1 T99N) as obstacles As the concentration of rigor kinesin increases the association time of mRNPs with MTs also increases. This suggests a mechanism that ensures that mRNPs stay associated with MTs longer in an obstacle rich environment compared to “naked” MTs Minus-end directed runs of mRNPs have a greater potential to pass the rigor kinesin as compared to plus-end directed runs of hairy mRNA. This may be due to dynein’s capacity to switch protofilaments (8) (9). 4Correlation analysis of mRNA5Microtubule end statistics6Conclusions (1) Novel assay to study mRNA transport termed RAT-TRAP was developed. (2) Direct encounter of single mRNPs with rigor kinesins (kinesin-1 T99N) was observed and, unlike purified unidirectional motor proteins (5) (10), mRNPs reverse often on encounter. Thus bidirectionality may be advantageous for staying associated with the microtubules for longer. (3) Minus-end and plus-end run length and velocity is tightly correlated for individual mRNPs whereas the correlation reduces when rigor kinesin is introduced as an obstacle on MTs. Taking into consideration the various models of bidirectional transport described in (11), we can eliminate recruitment dictated tug-of-war and motor specific regulatory complex as the possible models for these mRNPs. Bidirectional transport of mRNPs can be explained by the presence of a coordination complex or a mechanically coupled tug-of-war model. (4) Individual mRNPs tend to either pause or reverse on reaching the minus-end of MTs unlike encounter with rigor kinesin where they pause much less often. Hence the mechanisms associated with obstacle encounter and MTs minus-end encounter are different. (1) Holt CE, Bullock SL. Subcellular mRNA localization in animal cells and why it matters. Science. 326(5957): (2009). (2) Amrute-Nayak M, Bullock SL. Single-molecule assays reveal that RNA localization signals regulate dynein-dynactin copy number on individual transcript cargoes. Nat Cell Biol. 14, (2012). (3) Ross JL, Ali MY, Warshaw DM. Cargo transport: molecular motors navigate a complex cytoskeleton. Curr Opin Cell Biol. 20(1):41-47 (2008). (4) Gross SP. Hither and yon: a review of bi-directional microtubule-based transport. Phys Biol. 1(1-2):R1-11 (2004). (5) Telley IA, Bieling P, Surrey T. Obstacles on the microtubule reduce the processivity of Kinesin-1 in a minimal in vitro system and in cell extract. Biophys J. 96(8): (2009). (6) Wilkie GS, Davis I. Drosophila wingless and pair-rule transcripts localize apically by dynein-mediated transport of RNA particles. Cell 105, 209–219 (2001). (7) Vendra G, Hamilton RS & Davis I. Dynactin suppresses the retrograde movement of apically localized mRNA in Drosophila blastoderm embryos. RNA 13, 1860–1867 (2007). (8) Qiu W, Derr ND, Goodman BS, Villa E, Wu D, Shih W, Reck-Peterson SL. Dynein achieves processive motion using both stochastic and coordinated stepping. Nat Struct Mol Biol. 19(2): (2012). (9) DeWitt MA, Chang AY, Combs PA, Yildiz A. Cytoplasmic dynein moves through uncoordinated stepping of the AAA+ ring domains. Science. 335(6065): (2012). (10) Dixit R, Ross JL, Goldman YE, Holzbaur EL. Differential regulation of dynein and kinesin motor proteins by tau. Science. 319(5866): (2008). (11) Jolly AL, Gelfand VI. Bidirectional intracellular transport: utility and mechanism. Biochem Soc Trans. 39(5): (2011). Acknowledgements for reagents and advice: Andrew Carter, Manu Hegde, Carly Dix, Nick Barry and Yang Liu from MRC LMB, Cambridge; Thomas Surrey and Christian Duellberg from LRI London. References mRNPs have almost equal probability of pausing or reversing when the MT minus-end is reached; they seldom detach. This is different when compared to encounter of single mRNPs with rigor kinesin where they very rarely pause (Figure 2G). Mean run length and mean velocity are highly correlated on an individual mRNP basis Mean run length correlation may be due to different copy number of opposite polarity motors recruited to mRNPs Mean velocity correlation may be due to presence of non-motor MT interacting protein or motor copy number difference between mRNPs v Net motion and run length towards the minus and plus- end of MTs reduces as the concentration of rigor kinesin increases AB (A) (i) Full length mRNA with streptavidin (S1) aptamer linked to streptavidin (SA)-conjugated magnetic beads were incubated with embryo extracts. This allowed the capture of cargo–motor complexes, including those mRNPs containing the fluorescent RNA (inset) (ii) Following brief washing on a magnetic rack, motor complexes were released with biotin and injected into a flow cell containing immobilized Hilyte647-labelled MTs. (B) (i) Run lengths of minus-end and plus-end-directed runs of motile hairy wild-type mRNPs. (Note: Apical transport of hairy mRNA in the Drosophila blastoderm embryo is dependent on the dynein motor (6) and the dynactin complex (7).) (ii) Distributions of in vitro velocities for motile hairy wild-type mRNPs. In (i) and (ii) mean run length and velocity are calculated from gaussian distributions fitted to the data (dashed lines). C (C) (i) Quantification of net motion (unidirectional mRNPs in green), (ii) association times of Cy3 signals with MTs and (iii) reversal frequencies of motile hairy wild-type mRNPs. AC B D E (B) Quantification of net RNA motion of hairy mRNA with different concentration of rigor kinesin decorating MTs (as shown on X axis). Total number of mRNPs analyzed for 0nM, 10nM and 100nM rigor kinesin is 82, 77 and 60, respectively. (C) Distribution of minus-end and plus-end run length of hairy mRNA with different concentrations of rigor kinesin decorating the MTs (as shown on X axis). Number of runs analyzed given at the base of each bar. (D) Distribution of association times of hairy mRNA Cy3 signal with microtubules at different concentrations of rigor kinesin. (E) Reversal frequencies of hairy mRNA with different concentrations of rigor kinesin decorating the MTs. Correlation analysis of hairy mRNA with and without rigor kinesin on MTs. R2 represents the correlation coefficent and the closer the R2 is to 1 the more correlated the data set is and R2=1 represents a straight line. Each black circle represents an individual mRNP which is bidirectional and has more than 10 individual runs towards the minus or plus- end of MTs Direct observation of single hairy mRNP behaviour on reaching the minus-end of MTs FG (F) Kymograph shows hairy mRNA moving on MTs decorated with 10nM kinesin-1 T99N. It is evident from the kymograph on the left that the rigor kinesin can induce some reversals of mRNA. -/+ represents MT polarity. (G) Direct observation of encounters between single molecules of hairy mRNP and kinesin-1 T99N-mGFP at 10nM concentration. Total number of minus-end and plus-end encounters are provided as n value.