Probing cytoskeletal modulation of passive and active intracellular dynamics using nanobody-functionalized quantum dots

Abstract

The cytoplasm is a highly complex and heterogeneous medium that is structured by the cytoskeleton. How local transport depends on the heterogeneous organization and dynamics of F-actin and microtubules is poorly understood. Here we use a novel delivery and functionalization strategy to utilize quantum dots (QDs) as probes for active and passive intracellular transport. Rapid imaging of non-functionalized QDs reveals two populations with a 100-fold difference in diffusion constant, with the faster fraction increasing upon actin depolymerization. When nanobody-functionalized QDs are targeted to different kinesin motor proteins, their trajectories do not display strong actin-induced transverse displacements, as suggested previously. Only kinesin-1 displays subtle directional fluctuations, because the subset of microtubules used by this motor undergoes prominent undulations. Using actin-targeting agents reveals that F-actin suppresses most microtubule shape remodelling, rather than promoting it. These results demonstrate how the spatial heterogeneity of the cytoskeleton imposes large variations in non-equilibrium intracellular dynamics.

Actin filaments regulate microtubule growth at the centrosome

“…But these physical constraints may not be the only mechanism co-regulating the two networks at the centrosome. Noteworthy, these results add to pre-existing body of evidences showing that physical constraints imposed by actin filaments (Huber et al, 2015) can limit microtubule growth (Colin et al, 2018), microtubule's shape fluctuations (Brangwynne et al, 2006;Katrukha et al, 2017) and centrosome displacement (Piel et al, 2000). Interestingly, by contrast with previous descriptions of physical barriers blocking microtubule growth locally (Katrukha et al, 2017;Colin et al, 2018) our observations show that centrosomal actin filaments, by preventing microtubule growth at the organizing centre, affect the entire microtubule network throughout the cell.…”

“…The growth of microtubules can also be directed by actin-based structures (Kaverina et al, 1998;Théry et al, 2006;López et al, 2014). They can force the alignment of microtubules (Elie et al, 2015), resist their progression (Burnette et al, 2007), capture, bundle or stabilize them (Zhou et al, 2002;Hutchins & Wray, 2014), submit them to mechanical forces (Gupton et al, 2002;Fakhri et al, 2014;Robison et al, 2016) or define the limits in space into which they are confined (Katrukha et al, 2017). The actin-microtubule interplay mostly takes place at the cell periphery, because most actin filaments are nucleated at and reorganized into actin-based structures near the plasma membrane (Blanchoin et al, 2014).…”

Local actin nucleation tunes centrosomal microtubule nucleation during passage through mitosis

“…There are many ways in which the microtubule network can be influenced by actin filaments (Rodriguez et al, 2003;Coles & Bradke, 2015;Huber et al, 2015;Colin et al, 2018). Actin can direct the growth and alignment of microtubules (Kaverina et al, 1998;Thery et al, 2006;López et al, 2014;Elie et al, 2015), can change MT dynamics (Zhou et al, 2002;Hutchins & Wray, 2014) and can subject MTs to mechanical forces and physical constraints (Gupton et al, 2002;Brangwynne et al, 2006;Fakhri et al, 2014;Huber et al, 2015;Robison et al, 2016;Katrukha et al, 2017;Colin et al, 2018). These interactions occur along the length of microtubules and at their growing plus ends (Akhmanova & Steinmetz, 2015;Mohan & John, 2015), and are especially prevalent at the cell periphery (Waterman-Storer & Salmon, 1997;Wittmann et al, 2003), where the two filament systems converge in a crowded space.…”

F-actin patches associated with glutamatergic synapses control positioning of dendritic lysosomes

“…In another recent work we used quantum dots to study the heterogeneity of the cytoplasm and demonstrated that the F-actin-rich cellular cortex at the inner face of the plasma membrane is able to trap diffusive probes of a certain size (Katrukha et al, 2017). The presence of a dense actin mesh in dendrites might therefore influence the diffusion of molecules by confining local environments, and directly or indirectly influence organelle transport.…”

“…Interestingly, myosin V-induced anchoring of peroxisomes was sometimes observed along the axon, in dendrites and in the soma, suggesting that it occurs whenever cargoes with active myosin V encounter actin-rich regions (Janssen et al, 2017). In addition to active anchoring mechanisms, the presence of dense cytoskeletal structures and other organelles can create physical obstacles for cargo trafficking (Katrukha et al, 2017).…”