Using TIRFM we were able to visualize long trajectories of single cAR1 receptors in the basal plasma membrane that had been fluorescently tagged using the HaloTag technology. We found two populations of receptor molecules and characterized the types of diffusive behavior. We showed that 70% of the receptors are mobile and have directed motion with D=0.014 μm2
/s and v
= 0.18 ± 0.07 μm/s. No correlation was seen in the directionality of the receptor motion, but this can be attributed to the fact that we were concurrently observing few, well separated receptors. The directed motion is not a result of cell motility, since immobilizing the cell by disrupting the actin cytoskeleton with the actin depolymerizing drug Latrunculin A did not abolish the directed movement of cAR1 (). Disruption of the actin cytoskeleton resulted in an increase in cAR1 mobility, showing that actin cytoskeleton has an inhibitory effect on cAR1 mobility. Many reports have been published on the effect of the actin-based membrane cytoskeleton on GPCR mobility in the plasma membrane [35
]. One of the models proposed, the membrane-skeleton fence model, suggests that the GPCR is directly linked to the actin cytoskeleton and is temporarily confined. Another is the anchored-protein picket model where various transmembrane proteins are anchored and aligned along the membrane cytoskeleton, effectively forming rows of pickets. The hydrodynamic dragging effect of the pickets suppresses the movement of other molecules in the vicinity [38
]. No confinement was observed for cAR1 in the plasma membrane on the timescale we measured suggesting that cAR1 is slowed down by the ‘pickets’ on the actin cytoskeleton fence.
We were able to eliminate the directed movement of cAR1 in the basal plasma membrane on the timescale measured by destabilization of the microtubules. The microtubule effect was confirmed by our FRAP experiments with cAR1-eYFP. Microtubules-affecting drugs are very specific and FRAP on a plasma membrane marker showed that immobilizing cAR1 is not due to a change in the membrane viscosity as a side effect of benomyl. Disruption of cytoskeleton elements with the use of drugs can have either an inhibitory or a stimulatory effect in signaling depending on the system looked at [8
]. We investigated this and showed that when microtubules are destabilized, one of the first downstream cAMP signaling events, the jump of the Pleckstrin Homology domain (PH-GFP) of CRAC to the plasma membrane is prevented. We hypothesize that the microtubule-controlled mobility of cAR1 is necessary for cAR1 function, probably by enabling cAR1 to find his signaling partners in the plasma membrane. It has to be noted that it is possible that CRAC binding/transport could require microtubules, although this has not been documented before, independent of whether the receptor is mobile. However, resolving this issue would require uncoupling cAR1 from microtubules without disruption of the microtubules, necessitating more detailed knowledge of the biochemical link between cAR1 and microtubules.
We have shown that microtubules control the mobility of the majority of cAR1 molecules (70%) and that this is important for signaling. We also found a second population of molecules (30%) which we determined to be moving very slowly, since vibration in our set-up was negligible and there was movement to be seen on long timescales. This population may be unaffected by the cytoskeleton, either the actin network or microtubules, as the population was not affected by the drug latrunculin A and the degree of mobility is very close to that seen in benomyl-treated cells. To study the mobility of this population of receptors, imaging rates faster than are possible in our experimental set up are necessary Our imaging rate may be preventing us from discriminating the dynamics of the 30% immobile receptors from the benomyl affected 70% of mobile receptors. Thus, it is possible that the 30% of immobile receptors were not affected by the disruption of microtubules and hence not linked to microtubules, but to other signaling components. The plasma membrane contains diverse structures on nano-meso-scales (2–200 nm) with a variety of lifetimes. To see the structures at the lower limit of size and lifetime, one has to be able to image faster than the 30 ms timeframe applied in our study. In studies using colloidal gold particles in combination with Nomarski (differential interference contrast) or bright-field microscopy, imaging rates of 25 μs resolution can be reached [39
]. In our previous single-molecule study, where we showed that there are two receptor populations and that cAR1 mobility is polarized in the apical membrane of chemotaxing cells [14
], the diffusion coefficient was quite different compared to what we found in the basal membrane. This again supports the importance of the cytoskeleton in the mobility of cAR1, since the cortex is different for the apical membrane than that of the basal membrane in these rapid moving cells.
Though microtubules have been shown to control the lateral diffusion of plasma membrane receptors in other cell types, including neuronal cells [29
], the mechanism by which microtubules may affect mobility of cAR1 is unclear as there are only ~ 40 microtubules originating from the centrosome in Dictyostelium
cells. The actin network with its many crosslinks and connections with the plasma membrane has been widely described to control the mobility of membrane-localized proteins. It has recently been shown that microtubules have a similar type of network [40
]. Microtubules were found to attach to the basal cortex as a dynamic network with many microtubule-microtubule contacts in polarized epithelial cells. Several microtubule plus-end tip proteins, like adenomatous polyposis coli protein (APC) and end-binding protein 1 (EB1) were found to localize along the length of microtubules promoting microtubule-microtubule contacts and attaching the microtubules to the cell cortex along the length of the microtubules: microtubules were shown to grow over and pass APC puncta on the plasma membrane [40
] and these puncta could be part of a microdomain. Dictyostelium discoideum
have both APC and EB1 and DdEB1 was found to localize along the length of the microtubules [41
] indicating that a similar mechanism is possible.
cAR1, among other signaling proteins, was found in detergent-resistant membrane fractions indicating their organization in microdomains [42
]. Many signaling molecules have been found in detergent resistant membranes suggesting that these domains function as cell signaling platforms [43
]. We hypothesize that cAR1 is localized in microdomains and indirectly coupled to the network of microtubules via microtubule-binding proteins in the microdomains. These microdomains are tracking along microtubules via interaction of proteins in the domain and on the microtubules and thus causing the directed movement of cAR1. In mammalian cells it was found that Gα subunits bind directly to tubulin [44
]. In Dictyostelium
, the recently found homolog of the Fused kinase, called Tsunami, is localized in puncta at the periphery of the cell and in smaller spots along the length of microtubules in Dictyostelium
], similar to that found for APC in epithelial cells [40
]. It was suggested that Tsunami acts in association with microtubules and that it is important for polarization and chemotaxis in Dictyostelium
If cAR1 is diffusing in a small domain that tracks along the microtubule network, then cAR1 appears immobile upon microtubule disruption. Although cAR1 could still be diffusing in its domain, the receptors’ macroscopic (long-term) diffusion coefficients are now smaller than their microscopic (short-term) diffusion coefficients [39
]. A second possible explanation is that receptors are immobilized because of stabilization of the microtubule network. In MCF-7 cell, it has been shown that benomyl, at its half-maximal proliferation inhibitory concentration, strongly suppressed the rate and extent of growing and shortening excursions of individual microtubules without noticeably depolymerizing the microtubule network or decreasing the polymerized mass of tubulin [45
]. Furthermore, benomyl treatment caused an increase in the acetylation level of microtubules suggesting that it stabilizes microtubules. Although our study uses a benonmyl concentration that clearly depolymerized the microtubules originating from the centrosome (as shown in ), it is possible that the pieces of the suggested basal microtubule network were stabilized resulting in a fixed position of the domain or even a fixed position of cAR1 if there is a direct link.
Microtubules are of the utmost importance in signaling by trafficking signaling molecules to the signaling platform. The Ang II type 1 receptor (AT1
R) traffics into caveolae/lipid rafts together with Rac1 in a microtubule-dependent manner[9
]. Microtubules together with actin cytoskeleton restrict cAMP formation by regulating the localization and interaction of GPCR-Gs
-AC in caveolae/lipid rafts [8
]. Thus microtubules play a role in the localization of signaling proteins in caveolae/lipid microdomains. This is very important for the polarization of signaling in multiple processes including chemotaxis. In many cell types, including Dictyostelium
] there is an intriguing correlation between the position of the microtubule-organizing center (centrosome) and the direction of movement [16
]. Controlling and delivery of signaling molecules to microdomains could therefore be very important in creating a leading versus trailing edge during cell migration as was shown in neurons [29
We propose a mechanism in which cAR1 together with other signaling molecules are localized in microdomains, so called signalosomes, which laterally traffic along submembrane microtubules via microtubules-binding proteins in the domain. In addition, the microtubules also deliver signaling molecules to and from these signaling complexes. In this view the microtubules control the localization and the signaling of these signalosomes in the membrane and this is important in creating a leading versus trailing edge during chemotaxis. A similar mechanism could be essential in a wide variety of cell functions, including cytokinesis, and it will be important to characterize the components of the signalosomes and how they are regulated.