5. Transient transfection leads to highly heterogenous levels of
expression of the fluorophore (MCP-GFP-RBP protein).
Thus, there will be few cells which are extremely bright and
some that are extremely faint. Particle tracking in these cells is
extremely challenging due to low signal-to-noise ratio. Hence,
for single-molecule imaging select cells that have a high signalto-noise ratio for optimal single-particle tracking.
Acknowledgments
The authors would like to thank T. Lionnet (NYU) for helpful
discussion and all members of the Chao lab for their input and
the Facility for Advanced Imaging and Microscopy at the Friedrich
Miescher Institute for Biomedical Research for data acquisition and
image analysis support. Research in the Chao lab is funded by the
Novartis Research Foundation (J.A.C.), a Swiss National Science
Foundation (SNF) grant 31003A_182314 (J.A.C.), the
SNF-NCCR RNA & Disease (J.A.C.), and a Synapsis Foundation
grant 2017 CDA 01 (V.B.).
References
1. Tekotte H, Davis I (2002) Intracellular mRNA
localization: motors move messages. Trends
Genet 18:636–642
2. Gagnon JA, Mowry KL (2011) Molecular
motors: directing traffic during RNA localization. Crit Rev Biochem Mol Biol 46:229–239.
https://doi.org/10.3109/10409238.2011.
572861
3. Bullock SL, Nicol A, Gross SP, Zicha D (2006)
Guidance of bidirectional motor complexes by
mRNA cargoes through control of dynein
number
and
activity.
Curr
Biol
16:1447–1452. https://doi.org/10.1016/j.
cub.2006.05.055
4. Bullock SL (2011) Messengers, motors and
mysteries: sorting of eukaryotic mRNAs by
cytoskeletal transport. Biochem Soc Trans
39:1161–1165.
https://doi.org/10.1042/
BST0391161
5. Holt CE, Bullock SL (2009) Subcellular
mRNA localization in animal cells and why it
matters. Science 326:1212–1216. https://doi.
org/10.1126/science.1176488
6. St Johnston D (2005) Moving messages: the
intracellular localization of mRNAs. Nat Rev
Mol Cell Biol 6:363–375. https://doi.org/
10.1038/nrm1643
7. McCaffrey MW, Lindsay AJ (2012) Roles for
myosin Va in RNA transport and turnover.
Biochem Soc Trans 40:1416–1420. https://
doi.org/10.1042/BST20120172
8. Heym RG, Zimmermann D, Edelmann FT,
Israel L, O ¨ kten Z, Kovar DR, Niessing D
(2013) In vitro reconstitution of an mRNAtransport complex reveals mechanisms of
assembly and motor activation. J Cell Biol
203:971–984. https://doi.org/10.1083/jcb.
201302095
9. Kanai Y, Dohmae N, Hirokawa N (2004) Kinesin transports RNA: isolation and characterization of an RNA-transporting granule. Neuron
43:513–525. https://doi.org/10.1016/j.neu
ron.2004.07.022
10. Lazzaretti D, Bono F (2017) mRNA localization in metazoans: A structural perspective.
RNA Biol 14:1473–1484. https://doi.org/
10.1080/15476286.2017.1338231
11. Jansen R-P, Niessing D (2012) Assembly of
mRNA-protein complexes for directional
mRNA transport in eukaryotes--an overview.
Curr Protein Pept Sci 13:284–293. https://
doi.org/10.2174/138920312801619493
12. Coller J, Wickens M (2002) Tethered function
assays using 3
0 untranslated regions. Methods
26:142–150.
https://doi.org/10.1016/
S1046-2023(02)00016-6
13. Pillai RS, Artus CG, Filipowicz W (2004) Tethering of human Ago proteins to mRNA mimics
Tethering Assay for RNA Mobility
281
expression of the fluorophore (MCP-GFP-RBP protein).
Thus, there will be few cells which are extremely bright and
some that are extremely faint. Particle tracking in these cells is
extremely challenging due to low signal-to-noise ratio. Hence,
for single-molecule imaging select cells that have a high signalto-noise ratio for optimal single-particle tracking.
Acknowledgments
The authors would like to thank T. Lionnet (NYU) for helpful
discussion and all members of the Chao lab for their input and
the Facility for Advanced Imaging and Microscopy at the Friedrich
Miescher Institute for Biomedical Research for data acquisition and
image analysis support. Research in the Chao lab is funded by the
Novartis Research Foundation (J.A.C.), a Swiss National Science
Foundation (SNF) grant 31003A_182314 (J.A.C.), the
SNF-NCCR RNA & Disease (J.A.C.), and a Synapsis Foundation
grant 2017 CDA 01 (V.B.).
References
1. Tekotte H, Davis I (2002) Intracellular mRNA
localization: motors move messages. Trends
Genet 18:636–642
2. Gagnon JA, Mowry KL (2011) Molecular
motors: directing traffic during RNA localization. Crit Rev Biochem Mol Biol 46:229–239.
https://doi.org/10.3109/10409238.2011.
572861
3. Bullock SL, Nicol A, Gross SP, Zicha D (2006)
Guidance of bidirectional motor complexes by
mRNA cargoes through control of dynein
number
and
activity.
Curr
Biol
16:1447–1452. https://doi.org/10.1016/j.
cub.2006.05.055
4. Bullock SL (2011) Messengers, motors and
mysteries: sorting of eukaryotic mRNAs by
cytoskeletal transport. Biochem Soc Trans
39:1161–1165.
https://doi.org/10.1042/
BST0391161
5. Holt CE, Bullock SL (2009) Subcellular
mRNA localization in animal cells and why it
matters. Science 326:1212–1216. https://doi.
org/10.1126/science.1176488
6. St Johnston D (2005) Moving messages: the
intracellular localization of mRNAs. Nat Rev
Mol Cell Biol 6:363–375. https://doi.org/
10.1038/nrm1643
7. McCaffrey MW, Lindsay AJ (2012) Roles for
myosin Va in RNA transport and turnover.
Biochem Soc Trans 40:1416–1420. https://
doi.org/10.1042/BST20120172
8. Heym RG, Zimmermann D, Edelmann FT,
Israel L, O ¨ kten Z, Kovar DR, Niessing D
(2013) In vitro reconstitution of an mRNAtransport complex reveals mechanisms of
assembly and motor activation. J Cell Biol
203:971–984. https://doi.org/10.1083/jcb.
201302095
9. Kanai Y, Dohmae N, Hirokawa N (2004) Kinesin transports RNA: isolation and characterization of an RNA-transporting granule. Neuron
43:513–525. https://doi.org/10.1016/j.neu
ron.2004.07.022
10. Lazzaretti D, Bono F (2017) mRNA localization in metazoans: A structural perspective.
RNA Biol 14:1473–1484. https://doi.org/
10.1080/15476286.2017.1338231
11. Jansen R-P, Niessing D (2012) Assembly of
mRNA-protein complexes for directional
mRNA transport in eukaryotes--an overview.
Curr Protein Pept Sci 13:284–293. https://
doi.org/10.2174/138920312801619493
12. Coller J, Wickens M (2002) Tethered function
assays using 3
0 untranslated regions. Methods
26:142–150.
https://doi.org/10.1016/
S1046-2023(02)00016-6
13. Pillai RS, Artus CG, Filipowicz W (2004) Tethering of human Ago proteins to mRNA mimics
Tethering Assay for RNA Mobility
281