Microtubule damage shapes the acetylation gradient.

The properties of single microtubules within the microtubule network can be modulated through post-translational modifications (PTMs), including acetylation within the lumen of microtubules. To access the lumen, the enzymes could enter through the microtubule ends and at damage sites along the microtubule shaft. Here we show that the acetylation profile depends on damage sites, which can be caused by the motor protein kinesin-1. Indeed, the entry of the deacetylase HDAC6 into the microtubule lumen can be modulated by kinesin-1-induced damage sites. In contrast, activity of the microtubule acetylase αTAT1 is independent of kinesin-1-caused shaft damage. On a cellular level, our results show that microtubule acetylation distributes in an exponential gradient. This gradient results from tight regulation of microtubule (de)acetylation and scales with the size of the cells. The control of shaft damage represents a mechanism to regulate PTMs inside the microtubule by giving access to the lumen.

The effect of motor-induced shaft dynamics on microtubule stability and length.

Control of microtubule abundance, stability, and length is crucial to regulate intracellular transport as well as cell polarity and division. How microtubule stability depends on tubulin addition or removal at the dynamic ends is well studied. However, microtubule rescue, the event when a microtubule switches from shrinking to growing, occurs at tubulin exchange sites along the shaft. Molecular motors have recently been shown to promote such exchanges. Using a stochastic theoretical description, we study how microtubule stability and length depend on motor-induced tubulin exchange and thus rescue. Our theoretical description matches our in vitro experiments on microtubule dynamics in the presence of kinesin-1 molecular motors. Although the overall dynamics of a population of microtubules can be captured by an effective rescue rate, by assigning rescue to exchange sites, we reveal that the dynamics of individual microtubules within the population differ dramatically. Furthermore, we study in detail a transition from bounded to unbounded microtubule growth. Our results provide novel insights into how molecular motors imprint information of microtubule stability on the microtubule network.

Two-color in vitro assay to visualize and quantify microtubule shaft dynamics.

Microtubules are dynamic polymers where tubulin exchanges not only at the ends but also all along the microtubule shaft. In vitro reconstitutions are a vital approach to study microtubule tip dynamics, while direct observation of shaft dynamics is challenging. Here, we describe a dual-color in vitro assay to visualize microtubule shaft dynamics using purified, labeled bovine brain tubulin. With this assay, we can quantitatively address how proteins or small molecules impact the dynamics at the microtubule shaft. For complete details on the use and execution of this protocol, please refer to Andreu-Carbó et al. (2022).

Motor usage imprints microtubule stability along the shaft.

Tubulin dimers assemble into dynamic microtubules, which are used by molecular motors as tracks for intracellular transport. Organization and dynamics of the microtubule network are commonly thought to be regulated at the polymer ends, where tubulin dimers can be added or removed. Here, we show that molecular motors running on microtubules cause exchange of dimers along the shaft in vitro and in cells. These sites of dimer exchange act as rescue sites where depolymerizing microtubules stop shrinking and start re-growing. Consequently, the average length of microtubules increases depending on how frequently they are used as motor tracks. An increase of motor activity densifies the cellular microtubule network and enhances cell polarity. Running motors leave marks in the shaft, serving as traces of microtubule usage to organize the polarity landscape of the cell.

Phosphorylation of filamin A regulates chemokine receptor CCR2 recycling.

Proper endosomal trafficking of ligand-activated G-protein-coupled receptors (GPCRs) is essential to spatiotemporally tune their physiological responses. For the monocyte chemoattractant receptor 2 (CCR2B; one of two isoforms encoded by CCR2), endocytic recycling is important to sustain monocyte migration, whereas filamin A (FLNa) is essential for CCL2-induced monocyte migration. Here, we analyze the role of FLNa in the trafficking of CCR2B along the endocytic pathway. In FLNa-knockdown cells, activated CCR2B accumulated in enlarged EEA-1-positive endosomes, which exhibited slow movement and fast fluorescence recovery, suggesting an imbalance between receptor entry and exit rates. Utilizing super-resolution microscopy, we observed that FLNa-GFP, CCR2B and ß2-adrenergic receptor (ß2AR) were present in actin-enriched endosomal microdomains. Depletion of FLNa decreased CCR2B association with these microdomains and concomitantly delayed CCR2B endosomal traffic, without apparently affecting the number of microdomains. Interestingly, CCR2B and ß2AR signaling induced phosphorylation of FLNa at residue S2152, and this phosphorylation event was contributes to sustain receptor recycling. Thus, our data strongly suggest that CCR2B and ß2AR signals to FLNa to stimulate its endocytosis and recycling to the plasma membrane.