Intracellular transport is interspersed with frequent reversals in direction due to the presence of opposing kinesin and dynein motors on organelles that are carried as cargo. The cause and the mechanism of reversals are unknown, but are a key to understanding how cargos are delivered in a regulated manner to specific cellular locations. Unlike established single-motor biophysical assays, this problem requires understanding of the cooperative behavior of multiple interacting motors. Here we present measurements inside live Dictyostelium cells, in a cell extract and with purified motors to quantify such an ensemble function of motors. We show through precise motion analysis that reversals during endosome motion are caused by a tug-of-war between kinesin and dynein. Further, we use a combination of optical trap-based force measurements and Monte Carlo simulations to make the surprising discovery that endosome transport uses many (approximately four to eight) weak and detachment-prone dyneins in a tug-of-war against a single strong and tenacious kinesin. We elucidate how this clever choice of dissimilar motors and motor teams achieves net transport together with endosome fission, both of which are important in controlling the balance of endocytic sorting. To the best of our knowledge, this is a unique demonstration that dynein and kinesin function differently at the molecular level inside cells and of how this difference is used in a specific cellular process, namely endosome biogenesis. Our work may provide a platform to understand intracellular transport of a variety of organelles in terms of measurable quantities.asymmetric motor competition ͉ coordination of motors ͉ molecular motor dynein kinesin ͉ regulation of bidirectional motion ͉
In the bone marrow (BM), breast cancer cells (BCC) can survive in dormancy for decades as cancer stem cells (CSC), resurging as tertiary metastasis. The endosteal region where BCCs exist as CSCs poses a challenge to target them, mostly due to the coexistence of endogenous hematopoietic stem cells. This study addresses the early period of dormancy when BCCs enter BM at the perivascular region to begin the transition into CSCs, which we propose as the final step in dormancy. A two-step process comprises the Wnt-β-catenin pathway mediating BCC dedifferentiation into CSCs at the BM perivascular niche. At this site, BCCs responded to two types of mesenchymal stem cell (MSC)–released extracellular vesicles (EV) that may include exosomes. Early released EVs began the transition into cycling quiescence, DNA repair, and reorganization into distinct BCC subsets. After contact with breast cancer, the content of EVs changed (primed) to complete dedifferentiation into a more homogeneous population with CSC properties. BCC progenitors (Oct4alo), which are distant from CSCs in a hierarchical stratification, were sensitive to MSC EVs. Despite CSC function, Oct4alo BCCs expressed multipotent pathways similar to CSCs. Oct4alo BCCs dedifferentiated and colocalized with MSCs (murine and human BM) in vivo. Overall, these findings elucidate a mechanism of early dormancy at the BM perivascular region and provide evidence of epigenome reorganization as a potential new therapy for breast cancer. Significance: These findings describe how the initial process of dormancy and dedifferentiation of breast cancer cells at the bone marrow perivascular niche requires mesenchymal stem cell–derived exosomes, indicating a potential target for therapeutic intervention.
SignificanceThe liver secretes lipids in a controlled manner despite vast changes in its internal lipid content. This buffering function of the liver is essential for lipid/energy homeostasis, but its molecular and cellular mechanism is unknown. We show that motor protein kinesin transports lipid droplets (LDs) to the endoplasmic reticulum (ER) in liver cells, engineering ER−droplet contacts and supplying lipids to the ER for secretion as lipoprotein. However, when fasting induces massive lipid accumulation in liver, kinesin is removed from LDs, inhibiting lipid supply to the ER and homeostatically tempering lipid secretion from liver in a fasted state. Interestingly, reducing kinesin also blocks propagation of hepatitis-C virus inside liver cells, possibly because viral proteins cannot transfer from the ER to LDs.
We develop optical trapping methodology to precisely measure the force generated by motor-proteins on single organelles of unknown size in cell extract. Native motor-complexes can now be interrogated functionally, overcoming limitations of assays with purified motors coated on artificial beads. Forces, number and activity of kinesin-1 is measured on motile lipid droplets isolated from liver of normal and fasted rats to detect a correlation between metabolic state and kinesin-1 activity.
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