Plant hydraulics at the heart of plant, crops and ecosystem functions in the face of climate change.

Plant hydraulics is crucial for assessing the plants' capacity to extract and transport water from the soil up to their aerial organs. Along with their capacity to exchange water between plant compartments and regulate evaporation, hydraulic properties determine plant water relations, water status and susceptibility to pathogen attacks. Consequently, any variation in the hydraulic characteristics of plants is likely to significantly impact various mechanisms and processes related to plant growth, survival and production, as well as the risk of biotic attacks and forest fire behaviour. However, the integration of hydraulic traits into disciplines such as plant pathology, entomology, fire ecology or agriculture can be significantly improved. This review examines how plant hydraulics can provide new insights into our understanding of these processes, including modelling processes of vegetation dynamics, illuminating numerous perspectives for assessing the consequences of climate change on forest and agronomic systems, and addressing unanswered questions across multiple areas of knowledge.

Microclimate modulation: An overlooked mechanism influencing the impact of plant diversity on ecosystem functioning.

Changes in climate and biodiversity are widely recognized as primary global change drivers of ecosystem structure and functioning, also affecting ecosystem services provided to human populations. Increasing plant diversity not only enhances ecosystem functioning and stability but also mitigates climate change effects and buffers extreme weather conditions, yet the underlying mechanisms remain largely unclear. Recent studies have shown that plant diversity can mitigate climate change (e.g. reduce temperature fluctuations or drought through microclimatic effects) in different compartments of the focal ecosystem, which as such may contribute to the effect of plant diversity on ecosystem properties and functioning. However, these potential plant diversity-induced microclimate effects are not sufficiently understood. Here, we explored the consequences of climate modulation through microclimate modification by plant diversity for ecosystem functioning as a potential mechanism contributing to the widely documented biodiversity-ecosystem functioning (BEF) relationships, using a combination of theoretical and simulation approaches. We focused on a diverse set of response variables at various levels of integration ranging from ecosystem-level carbon exchange to soil enzyme activity, including population dynamics and the activity of specific organisms. Here, we demonstrated that a vegetation layer composed of many plant species has the potential to influence ecosystem functioning and stability through the modification of microclimatic conditions, thus mitigating the negative impacts of climate extremes on ecosystem functioning. Integrating microclimatic processes (e.g. temperature, humidity and light modulation) as a mechanism contributing to the BEF relationships is a promising avenue to improve our understanding of the effects of climate change on ecosystem functioning and to better predict future ecosystem structure, functioning and services. In addition, microclimate management and monitoring should be seen as a potential tool by practitioners to adapt ecosystems to climate change.

Functionally distinct tree species support long-term productivity in extreme environments.

Despite evidence of a positive effect of functional diversity on ecosystem productivity, the importance of functionally distinct species (i.e. species that display an original combination of traits) is poorly understood. To investigate how distinct species affect ecosystem productivity, we used a forest-gap model to simulate realistic temperate forest successions along an environmental gradient and measured ecosystem productivity at the end of the successional trajectories. We performed 10 560 simulations with different sets and numbers of species, bearing either distinct or indistinct functional traits, and compared them to random assemblages, to mimic the consequences of a regional loss of species. Long-term ecosystem productivity dropped when distinct species were lost first from the regional pool of species, under the harshest environmental conditions. On the contrary, productivity was more dependent on ordinary species in milder environments. Our findings show that species functional distinctiveness, integrating multiple trait dimensions, can capture species-specific effects on ecosystem productivity. In a context of an environmentally changing world, they highlight the need to investigate the role of distinct species in sustaining ecosystem processes, particularly in extreme environmental conditions.

Small and slow is safe: On the drought tolerance of tropical tree species.

Understanding how evolutionary history and the coordination between trait trade-off axes shape the drought tolerance of trees is crucial to predict forest dynamics under climate change. Here, we compiled traits related to drought tolerance and the fast-slow and stature-recruitment trade-off axes in 601 tropical woody species to explore their covariations and phylogenetic signals. We found that xylem resistance to embolism (P50) determines the risk of hydraulic failure, while the functional significance of leaf turgor loss point (TLP) relies on its coordination with water use strategies. P50 and TLP exhibit weak phylogenetic signals and substantial variation within genera. TLP is closely associated with the fast-slow trait axis: slow species maintain leaf functioning under higher water stress. P50 is associated with both the fast-slow and stature-recruitment trait axes: slow and small species exhibit more resistant xylem. Lower leaf phosphorus concentration is associated with more resistant xylem, which suggests a (nutrient and drought) stress-tolerance syndrome in the tropics. Overall, our results imply that (1) drought tolerance is under strong selective pressure in tropical forests, and TLP and P50 result from the repeated evolutionary adaptation of closely related taxa, and (2) drought tolerance is coordinated with the ecological strategies governing tropical forest demography. These findings provide a physiological basis to interpret the drought-induced shift toward slow-growing, smaller, denser-wooded trees observed in the tropics, with implications for forest restoration programmes.

Mib1 prevents Notch Cis-inhibition to defer differentiation and preserve neuroepithelial integrity during neural delamination.

The vertebrate neuroepithelium is composed of elongated progenitors whose reciprocal attachments ensure the continuity of the ventricular wall. As progenitors commit to differentiation, they translocate their nucleus basally and eventually withdraw their apical endfoot from the ventricular surface. However, the mechanisms allowing this delamination process to take place while preserving the integrity of the neuroepithelial tissue are still unclear. Here, we show that Notch signaling, which is classically associated with an undifferentiated state, remains active in prospective neurons until they delaminate. During this transition period, prospective neurons rapidly reduce their apical surface and only later down-regulate N-Cadherin levels. Upon Notch blockade, nascent neurons disassemble their junctions but fail to reduce their apical surface. This disrupted sequence weakens the junctional network and eventually leads to breaches in the ventricular wall. We also provide evidence that the Notch ligand Delta-like 1 (Dll1) promotes differentiation by reducing Notch signaling through a Cis-inhibition mechanism. However, during the delamination process, the ubiquitin ligase Mindbomb1 (Mib1) transiently blocks this Cis-inhibition and sustains Notch activity to defer differentiation. We propose that the fine-tuned balance between Notch Trans-activation and Cis-inhibition allows neuroepithelial cells to seamlessly delaminate from the ventricular wall as they commit to differentiation.

Regulation of the Expression, Oligomerisation and Signaling of the Inhibitory Receptor CLEC12A by Cysteine Residues in the Stalk Region.

CLEC12A is a myeloid inhibitory receptor that negatively regulates inflammation in mouse models of autoimmune and autoinflammatory arthritis. Reduced CLEC12A expression enhances myeloid cell activation and inflammation in CLEC12A knock-out mice with collagen antibody-induced or gout-like arthritis. Similarly to other C-type lectin receptors, CLEC12A harbours a stalk domain between its ligand binding and transmembrane domains. While it is presumed that the cysteines in the stalk domain have multimerisation properties, their role in CLEC12A expression and/or signaling remain unknown. We thus used site-directed mutagenesis to determine whether the stalk domain cysteines play a role in CLEC12A expression, internalisation, oligomerisation, and/or signaling. Mutation of C118 blocks CLEC12A transport through the secretory pathway diminishing its cell-surface expression. In contrast, mutating C130 does not affect CLEC12A cell-surface expression but increases its oligomerisation, inducing ligand-independent phosphorylation of the receptor. Moreover, we provide evidence that CLEC12A dimerisation is regulated in a redox-dependent manner. We also show that antibody-induced CLEC12A cross-linking induces flotillin oligomerisation in insoluble membrane domains in which CLEC12A signals. Taken together, these data indicate that the stalk cysteines in CLEC12A differentially modulate this inhibitory receptor's expression, oligomerisation and signaling, suggestive of the regulation of CLEC12A in a redox-dependent manner during inflammation.

Climate-associated genetic variation in Fagus sylvatica and potential responses to climate change in the French Alps.

Local adaptation patterns have been found in many plants and animals, highlighting the genetic heterogeneity of species along their range of distribution. In the next decades, global warming is predicted to induce a change in the selective pressures that drive this adaptive variation, forcing a reshuffling of the underlying adaptive allele distributions. For species with low dispersion capacity and long generation time such as trees, the rapidity of the change could impede the migration of beneficial alleles and lower their capacity to track the changing environment. Identifying the main selective pressures driving the adaptive genetic variation is thus necessary when investigating species capacity to respond to global warming. In this study, we investigate the adaptive landscape of Fagus sylvatica along a gradient of populations in the French Alps. Using a double-digest restriction-site-associated DNA (ddRAD) sequencing approach, we identified 7,000 SNPs from 570 individuals across 36 different sites. A redundancy analysis (RDA)-derived method allowed us to identify several SNPs that were strongly associated with climatic gradients; moreover, we defined the primary selective gradients along the natural populations of F. sylvatica in the Alps. Strong effects of elevation and humidity, which contrast north-western and south-eastern site, were found and were believed to be important drivers of genetic adaptation. Finally, simulations of future genetic landscapes that used these findings allowed identifying populations at risk for F. sylvatica in the Alps, which could be helpful for future management plans.

A Far-Red Emitting Fluorescent Chemogenetic Reporter for In†Vivo Molecular Imaging.

Far-red emitting fluorescent labels are highly desirable for spectral multiplexing and deep tissue imaging. Here, we describe the generation of frFAST (far-red Fluorescence Activating and absorption Shifting Tag), a 14-kDa monomeric protein that forms a bright far-red fluorescent assembly with (4-hydroxy-3-methoxy-phenyl)allylidene rhodanine (HPAR-3OM). As HPAR-3OM is essentially non-fluorescent in solution and in cells, frFAST can be imaged with high contrast in presence of free HPAR-3OM, which allowed the rapid and efficient imaging of frFAST fusions in live cells, zebrafish embryo/larvae, and chicken embryos. Beyond enabling the genetic encoding of far-red fluorescence, frFAST allowed the design of a far-red chemogenetic reporter of protein-protein interactions, demonstrating its great potential for the design of innovative far-red emitting biosensors.

Loss of the canonical spindle orientation function in the Pins/LGN homolog AGS3.

In many cell types, mitotic spindle orientation relies on the canonical "LGN complex" composed of Pins/LGN, Mud/NuMA, and Gαi subunits. Membrane localization of this complex recruits motor force generators that pull on astral microtubules to orient the spindle. Drosophila Pins shares highly conserved functional domains with its two vertebrate homologs LGN and AGS3. Whereas the role of Pins and LGN in oriented divisions is extensively documented, involvement of AGS3 remains controversial. Here, we show that AGS3 is not required for planar divisions of neural progenitors in the mouse neocortex. AGS3 is not recruited to the cell cortex and does not rescue LGN loss of function. Despite conserved interactions with NuMA and Gαiin vitro, comparison of LGN and AGS3 functional domains in vivo reveals unexpected differences in the ability of these interactions to mediate spindle orientation functions. Finally, we find that Drosophila Pins is unable to substitute for LGN loss of function in vertebrates, highlighting that species-specific modulations of the interactions between components of the Pins/LGN complex are crucial in vivo for spindle orientation.

Regulation of mitotic spindle orientation: an integrated view.

Mitotic spindle orientation is essential for cell fate decisions, epithelial maintenance, and tissue morphogenesis. In most animal cell types, the dynein motor complex is anchored at the cell cortex and exerts pulling forces on astral microtubules to position the spindle. Early studies identified the evolutionarily conserved Gαi/LGN/NuMA complex as a key regulator that polarizes cortical force generators. In recent years, a combination of genetics, biochemistry, modeling, and live imaging has contributed to decipher the mechanisms of spindle orientation. Here, we highlight the dynamic nature of the assembly of this complex and discuss the molecular regulation of its localization. Remarkably, a number of LGN-independent mechanisms were described recently, whereas NuMA remains central in most pathways involved in recruiting force generators at the cell cortex. We also describe the emerging role of the actin cortex in spindle orientation and discuss how dynamic astral microtubule formation is involved. We further give an overview on instructive external signals that control spindle orientation in tissues. Finally, we discuss the influence of cell geometry and mechanical forces on spindle orientation.

The limited contribution of large trees to annual biomass production in an old-growth tropical forest.

Although the importance of large trees regarding biodiversity and carbon stock in old-growth forests is undeniable, their annual contribution to biomass production and carbon uptake remains poorly studied at the stand level. To clarify the role of large trees in biomass production, we used data of tree growth, mortality, and recruitment monitored during 20 yr in 10 4-ha plots in a species-rich tropical forest (Central African Republic). Using a random block design, three different silvicultural treatments, control, logged, and logged + thinned, were applied in the 10 plots. Annual biomass gains and losses were analyzed in relation to the relative biomass abundance of large trees and by tree size classes using a spatial bootstrap procedure. Although large trees had high individual growth rates and constituted a substantial amount of biomass, stand-level biomass production decreased with the abundance of large trees in all treatments and plots. The contribution of large trees to annual stand-level biomass production appeared limited in comparison to that of small trees. This pattern did not only originate from differences in abundance of small vs. large trees or differences in initial biomass stocks among tree size classes, but also from a reduced relative growth rate of large trees and a relatively constant mortality rate among tree size classes. In a context in which large trees are increasingly gaining attention as being a valuable and a key structural characteristic of natural forests, the present study brought key insights to better gauge the relatively limited role of large trees in annual stand-level biomass production. In terms of carbon uptake, these results suggest, as already demonstrated, a low net carbon uptake of old-growth forests in comparison to that of logged forests. Tropical forests that reach a successional stage with relatively high density of large trees progressively cease to be carbon sinks as large trees contribute sparsely or even negatively to the carbon uptake at the stand level.

Bifunctional fusion proteins containing the sequence of the bradykinin homologue maximakinin: activities at the rat bradykinin B2 receptor.

To support bradykinin (BK) B2 receptor (B2R) detection and therapeutic stimulation, we developed and characterized fusion proteins consisting of the BK homolog maximakinin (MK), or variants, positioned at the C-terminus of functional proteins (enhanced green fluorescent protein (EGFP), the peroxidase APEX2, or human serum albumin (HSA)). EGFP-MK loses its reactivity with anti-BK antibodies and molecular mass as it progresses in the endosomal tract of cells expressing rat B2Rs (immunoblots, epifluorescence microscopy). APEX2-(NG)15-MK is a bona fide agonist of the rat, but not of the human B2R (calcium and c-Fos signaling) and is compatible with the cytochemistry reagent TrueBlue (microscopy), a luminol-based reagent, or 3,3',5,5'-tetramethylbenzidine (luminescence or colourimetric B2R detection, cell well plate format). APEX2-(NG)15-MK is a non-isotopic ligand suitable for drug discovery via binding competition. Affinity-purified secreted forms of HSA fused with peptides possessing the C-terminal MK or BK sequence failed to stimulate the rat B2R in the concentration range of 50-600 nmol/L. However, the non-secreted construction myc-HSA-MK is a B2R agonist, indicating that protein denaturation made the C-terminal sequence available for receptor binding. Fusion protein ligands of the B2R are stable but subjected to slow intracellular inactivation, strong species specificity, and possible steric hindrance between the receptor and large proteins.

Detection and tracking of overlapping cell nuclei for large scale mitosis analyses.

BACKGROUND: Cell culture on printed micropatterns slides combined with automated fluorescent microscopy allows for extraction of tens of thousands of videos of small isolated growing cell clusters. The analysis of such large dataset in space and time is of great interest to the community in order to identify factors involved in cell growth, cell division or tissue formation by testing multiples conditions. However, cells growing on a micropattern tend to be tightly packed and to overlap with each other. Consequently, image analysis of those large dynamic datasets with no possible human intervention has proven impossible using state of the art automated cell detection methods. RESULTS: Here, we propose a fully automated image analysis approach to estimate the number, the location and the shape of each cell nucleus, in clusters at high throughput. The method is based on a robust fit of Gaussian mixture models with two and three components on each frame followed by an analysis over time of the fitting residual and two other relevant features. We use it to identify with high precision the very first frame containing three cells. This allows in our case to measure a cell division angle on each video and to construct division angle distributions for each tested condition. We demonstrate the accuracy of our method by validating it against manual annotation on about 4000 videos of cell clusters. CONCLUSIONS: The proposed approach enables the high throughput analysis of video sequences of isolated cell clusters obtained using micropatterns. It relies only on two parameters that can be set robustly as they reduce to the average cell size and intensity.

Explanatory ecological factors for the persistence of desiccation-sensitive seeds in transient soil seed banks: Quercus ilex as a case study.

BACKGROUND AND AIMS: Dominant tree species in northern temperate forests, for example oak and beech, produce desiccation-sensitive seeds. Despite the potentially major influence of this functional trait on the regeneration and distribution of species under climate change, little is currently known about the ecological determinants of the persistence of desiccation-sensitive seeds in transient soil seed banks. Knowing which key climatic and microsite factors favour seed survival will help define the regeneration niche for species whose seeds display extreme sensitivity to environmental stress METHODS: Using the Mediterranean Holm oak (Quercus ilex) forest as a model system, an in situ time-course monitoring of seed water status and viability was performed during the unfavourable winter season in two years with contrasting rainfall, at an instrumented site with detailed climate records. In parallel, the characteristics of the microhabitat and their influence on the post-winter water status and viability of seeds were investigated in a regional survey of 33 woodlands representative of the French distribution of the species. KEY RESULTS: Time-course monitoring of seed water status in natural conditions confirmed that in situ desiccation is the main abiotic cause of mortality in winter. Critical water contents could be reached in a few days during drought spells. Seed dehydration rates were satisfactorily estimated using integrative climate proxies including vapour pressure deficit and potential evapotranspiration. Seed water status was therefore determined by the balance between water uptake after a rainfall event and water loss during dry periods. Structural equation modelling of microhabitat factors highlighted the major influence of canopy openness and resulting incident radiation on the ground. CONCLUSIONS: This study provides part of the knowledge required to implement species distribution models which incorporate their regeneration niche. It is an important step forward in evaluating the ecological consequences of increasing winter drought and environmental filtering due to climate change on the regeneration of the most dominant Mediterranean tree species.

Predictive Spatiotemporal Manipulation of Signaling Perturbations Using Optogenetics.

Recently developed optogenetic methods promise to revolutionize cell biology by allowing signaling perturbations to be controlled in space and time with light. However, a quantitative analysis of the relationship between a custom-defined illumination pattern and the resulting signaling perturbation is lacking. Here, we characterize the biophysical processes governing the localized recruitment of the Cryptochrome CRY2 to its membrane-anchored CIBN partner. We develop a quantitative framework and present simple procedures that enable predictive manipulation of protein distributions on the plasma membrane with a spatial resolution of 5 µm. We show that protein gradients of desired levels can be established in a few tens of seconds and then steadily maintained. These protein gradients can be entirely relocalized in a few minutes. We apply our approach to the control of the Cdc42 Rho GTPase activity. By inducing strong localized signaling perturbation, we are able to monitor the initiation of cell polarity and migration with a remarkable reproducibility despite cell-to-cell variability.

Multicolor two-photon tissue imaging by wavelength mixing.

We achieve simultaneous two-photon excitation of three chromophores with distinct absorption spectra using synchronized pulses from a femtosecond laser and an optical parametric oscillator. The two beams generate separate multiphoton processes, and their spatiotemporal overlap provides an additional two-photon excitation route, with submicrometer overlay of the color channels. We report volume and live multicolor imaging of 'Brainbow'-labeled tissues as well as simultaneous three-color fluorescence and third-harmonic imaging of fly embryos.

Temporal stability in forest productivity increases with tree diversity due to asynchrony in species dynamics.

Theory predicts a positive relationship between biodiversity and stability in ecosystem properties, while diversity is expected to have a negative impact on stability at the species level. We used virtual experiments based on a dynamic simulation model to test for the diversity-stability relationship and its underlying mechanisms in Central European forests. First our results show that variability in productivity between stands differing in species composition decreases as species richness and functional diversity increase. Second we show temporal stability increases with increasing diversity due to compensatory dynamics across species, supporting the biodiversity insurance hypothesis. We demonstrate that this pattern is mainly driven by the asynchrony of species responses to small disturbances rather than to environmental fluctuations, and is only weakly affected by the net biodiversity effect on productivity. Furthermore, our results suggest that compensatory dynamics between species may enhance ecosystem stability through an optimisation of canopy occupancy by coexisting species.

Tree inventory data from permanent plots in French forest reserves.

We present a data set resulting from the first round of a national monitoring program of forest reserves. It contains 9538 permanent plots, distributed across 111 study sites in mainland France (including Corsica). Notably focusing on dead wood measurement, this protocol has primarily been applied in strict forest reserves and special nature reserves (sensu Bollmann & Braunisch 2013), with 68% (6494) of the plots being currently located in strict forest reserves (unmanaged) and 24.7% (2363 plots) in forests unmanaged for at least 50 years. Sites cover a large variety of ecological conditions, from lowland to subalpine forests, but with an underrepresentation of Mediterranean forests (Table 1). The protocol assesses all the stages of a tree's life cycle, from seedling to decomposed lying dead wood. On each plot, a combination of three sampling techniques was used: (1) fixed-area inventory for regeneration, standing dead trees, living trees, and coarse woody debris (CWD) with diameter over 30 cm; (2) transect lines for CWD with diameter <30 cm; and (3) fixed-angle plot method for living trees with diameter at breast height (DBH) >30 cm (using a relascopic angle of 3%). Measurements include exact tree location (azimuth, distance), species, diameter(s), tree-related microhabitats, decay stage and bark cover, and seedling cover. With ongoing climate change, the program network can also provide important information to monitor changes in forest ecosystems. It can also be used as forest management monitoring or conservation status assessment. These data are freely available for noncommercial scientific use (Creative Commons Attribution 4.0 CC BY SA 4.0) with attribution, and this paper must be cited if this material is reused.

Can mixing Quercus robur and Quercus petraea with Pinus sylvestris compensate for productivity losses due to climate change?

The climate change scenarios RCP 4.5 and RCP 8.5, with a representative concentration pathway for stabilization of radiative forcing of 4.5 W m-2 and 8.5 W m-2 by 2100, respectively, predict an increase in temperature of 1-4.5° Celsius for Europe and a simultaneous shift in precipitation patterns leading to increased drought frequency and severity. The negative consequences of such changes on tree growth on dry sites or at the dry end of a tree species distribution are well-known, but rarely quantified across large gradients. In this study, the growth of Quercus robur and Quercus petraea (Q. spp.) and Pinus sylvestris in pure and mixed stands was predicted for a historical scenario and the two climate change scenarios RCP 4.5 and RCP 8.5 using the individual tree growth model PrognAus. Predictions were made along an ecological gradient ranging from current mean annual temperatures of 5.5-11.4 °C and with mean annual precipitation sums of 586-929 mm. Initial data for the simulation consisted of 23 triplets established in pure and mixed stands of Q. spp. and P. sylvestris. After doing the simulations until 2100, we fitted a linear mixed model using the predicted volume in the year 2100 as response variable to describe the general trends in the simulation results. Productivity decreased for both Q. spp. and P. sylvestris with increasing temperature, and more so, for the warmer sites of the gradient. P. sylvestris is the more productive tree species in the current climate scenario, but the competitive advantage shifts to Q. spp., which is capable to endure very high negative water potentials, for the more severe climate change scenario. The Q. spp.-P. sylvestris mixture presents an intermediate resilience to increased scenario severity. Enrichment of P. sylvestris stands by creating mixtures with Q. spp., but not the opposite, might be a right silvicultural adaptive strategy, especially at lower latitudes. Tree species mixing can only partly compensate productivity losses due to climate change. This may, however, be possible in combination with other silvicultural adaptation strategies, such as thinning and uneven-aged management.

Inhibitory effects of cytoskeleton disrupting drugs and GDP-locked Rab mutants on bradykinin B2 receptor cycling.

The bradykinin (BK) B2 receptor (B2R) is G protein coupled and phosphorylated upon agonist stimulation; its endocytosis and recycling are documented. We assessed the effect of drugs that affect the cytoskeleton on B2R cycling. These drugs were targeted to tubulin (paclitaxel, or the novel combretastatin A-4 mimetic 3,4,5-trimethoxyphenyl-4-(2-oxoimidazolidin-1-yl)benzenesulfonate [IMZ-602]) and actin (cytochalasin D). Tubulin ligands did not alter agonist-induced receptor endocytosis, as shown using antibodies reactive with myc-tagged B2Rs (microscopy, cytofluorometry), but rather reduced the progression of the ligand-receptor-ß-arrestin complex from the cell periphery to the interior. The 3 fluorescent probes of this complex (B2R-green fluorescent protein [B2R-GFP], the fluorescent agonist fluorescein-5-thiocarbamoyl-D-Arg-[Hyp³, Igl5, Oic7, Igl8]-BK and ß-arrestin2-GFP) were condensed in punctuate structures that remained close to the cell surface in the presence of IMZ-602. Cytochalasin D selectively inhibited the recycling of endocytosed B2R-GFP (B2R-GFP imaging, [³H]BK binding). Dominant negative (GDP-locked)-Rab5 and -Rab11 reproduced the effects of inhibitors of tubulin and actin, respectively, on the cycling of B2R-GFP. GDP-locked-Rab4 also inhibited B2R-GFP recycling to the cell surface. Consistent with the displacement of cargo along specific cytoskeletal elements, Rab5-associated progression of the endocytosed BK B2R follows microtubules toward their (-) end, while its recycling progresses along actin fibers to the cell surface. However, tubulin ligands do not suppress the tested desensitization or resensitization mechanisms of the B2R.