Abstract
Molecular motor proteins are responsible for long-range transport of vesicles and organelles. Recent works have elucidated the richness of the transport complex, with multiple teams of similar and dissimilar motors and their cofactors attached to individual cargoes. The interaction among these different proteins, and with the microtubules along which they translocate, results in the intricate patterns of cargo transport observed in cells. High-precision and high-bandwidth measurements are required to capture the dynamics of these interactions, yet the crowdedness in the cell necessitates performing such measurements in vitro. Here, we show that endogenous cargoes, lipid droplets purified from Drosophila embryos, can be used to perform high-precision and high-bandwidth optical trapping experiments to study motor regulation in vitro. Purified droplets have constituents of the endogenous transport complex attached to them and exhibit long-range motility. A novel method to determine the quality of the droplets for high-resolution measurements in an optical trap showed that they compare well with plastic beads in terms of roundness, homogeneity, position sensitivity, and trapping stiffness. Using high-resolution and high-bandwidth position measurements, we demonstrate that we can follow the series of binding and unbinding events that lead to the onset of active transport.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1182-1191 |
| Number of pages | 10 |
| Journal | Biophysical Journal |
| Volume | 105 |
| Issue number | 5 |
| DOIs | |
| State | Published - Sep 3 2013 |
Fingerprint
ASJC Scopus subject areas
- Biophysics
Cite this
Lipid droplets purified from drosophila embryos as an endogenous handle for precise motor transport measurements. / Bartsch, Tobias F.; Longoria, Rafael A.; Florin, Ernst Ludwig; Shubeita, George.
In: Biophysical Journal, Vol. 105, No. 5, 03.09.2013, p. 1182-1191.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Lipid droplets purified from drosophila embryos as an endogenous handle for precise motor transport measurements
AU - Bartsch, Tobias F.
AU - Longoria, Rafael A.
AU - Florin, Ernst Ludwig
AU - Shubeita, George
PY - 2013/9/3
Y1 - 2013/9/3
N2 - Molecular motor proteins are responsible for long-range transport of vesicles and organelles. Recent works have elucidated the richness of the transport complex, with multiple teams of similar and dissimilar motors and their cofactors attached to individual cargoes. The interaction among these different proteins, and with the microtubules along which they translocate, results in the intricate patterns of cargo transport observed in cells. High-precision and high-bandwidth measurements are required to capture the dynamics of these interactions, yet the crowdedness in the cell necessitates performing such measurements in vitro. Here, we show that endogenous cargoes, lipid droplets purified from Drosophila embryos, can be used to perform high-precision and high-bandwidth optical trapping experiments to study motor regulation in vitro. Purified droplets have constituents of the endogenous transport complex attached to them and exhibit long-range motility. A novel method to determine the quality of the droplets for high-resolution measurements in an optical trap showed that they compare well with plastic beads in terms of roundness, homogeneity, position sensitivity, and trapping stiffness. Using high-resolution and high-bandwidth position measurements, we demonstrate that we can follow the series of binding and unbinding events that lead to the onset of active transport.
AB - Molecular motor proteins are responsible for long-range transport of vesicles and organelles. Recent works have elucidated the richness of the transport complex, with multiple teams of similar and dissimilar motors and their cofactors attached to individual cargoes. The interaction among these different proteins, and with the microtubules along which they translocate, results in the intricate patterns of cargo transport observed in cells. High-precision and high-bandwidth measurements are required to capture the dynamics of these interactions, yet the crowdedness in the cell necessitates performing such measurements in vitro. Here, we show that endogenous cargoes, lipid droplets purified from Drosophila embryos, can be used to perform high-precision and high-bandwidth optical trapping experiments to study motor regulation in vitro. Purified droplets have constituents of the endogenous transport complex attached to them and exhibit long-range motility. A novel method to determine the quality of the droplets for high-resolution measurements in an optical trap showed that they compare well with plastic beads in terms of roundness, homogeneity, position sensitivity, and trapping stiffness. Using high-resolution and high-bandwidth position measurements, we demonstrate that we can follow the series of binding and unbinding events that lead to the onset of active transport.
UR - http://www.scopus.com/inward/record.url?scp=84883623796&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84883623796&partnerID=8YFLogxK
U2 - 10.1016/j.bpj.2013.07.026
DO - 10.1016/j.bpj.2013.07.026
M3 - Article
VL - 105
SP - 1182
EP - 1191
JO - Biophysical Journal
JF - Biophysical Journal
SN - 0006-3495
IS - 5
ER -