Franck Perez

Rac1 and Cdc42 Capture Microtubules through IQGAP1 and CLIP-170

Linkage of microtubules to special cortical regions is essential for cell polarization. CLIP-170 binds to the growing ends of microtubules and plays pivotal roles in orientation. We have found that IQGAP1, an effector of Rac1 and Cdc42, interacts with CLIP-170. In Vero fibroblasts, IQGAP1 localizes at the polarized leading edge. Expression of carboxy-terminal fragment of IQGAP1, which includes the CLIP-170 binding region, delocalizes GFP-CLIP-170 from the tips of microtubules and alters the microtubule array. Activated Rac1/Cdc42, IQGAP1, and CLIP-170 form a tripartite complex. Furthermore, expression of an IQGAP1 mutant defective in Rac1/Cdc42 binding induces multiple leading edges. These results indicate that Rac1/Cdc42 marks special cortical spots where the IQGAP1 and CLIP-170 complex is targeted, leading to a polarized microtubule array and cell polarization.

CLIP-170 Highlights Growing Microtubule Ends In Vivo

A chimera with the green fluorescent protein (GFP) has been constructed to visualize the dynamic properties of the endosome-microtubule linker protein CLIP170 (GFP-CLIP170). GFP-CLIP170 binds in stretches along a subset of microtubule ends. These fluorescent stretches appear to move with the growing tips of microtubules at 0.15-0.4 microm/s, comparable to microtubule elongation in vivo. Analysis of speckles along dynamic GFP-CLIP170 stretches suggests that CLIP170 treadmills on growing microtubule ends, rather than being continuously transported toward these ends. Drugs affecting microtubule dynamics rapidly inhibit movement of GFP-CLIP170 dashes. We propose that GFP-CLIP170 highlights growing microtubule ends by specifically recognizing the structure of a segment of newly polymerized tubulin.

ESCRT Machinery Is Required for Plasma Membrane Repair

Introduction Plasma membrane damage can result from numerous threats, including mechanical stress or biochemical agents such as pore-forming toxins. Different mechanisms for plasma membrane repair have been described in a variety of cellular models, including patching with endomembranes, endocytosis, and extracellular budding. We found that the endosomal sorting complex required for transport (ESCRT), which is implicated in numerous membrane fission events (such as during cytokinesis or for the budding of several viruses) was also required for the rapid closure of small wounds made at the plasma membrane. ESCRT recruitment mediates pinching out of wounded plasma membrane. (A) Cells expressing the ESCRT subunit CHMP4B-EGFP and wounded (arrow) in the presence of propidium iodide (PI) were observed by means of fluorescence imaging. (B) Model for ESCRT-mediated detection and shedding of wounded plasma membrane. Methods We used micropipettes, detergents, pore-forming toxins, and laser wounding to damage the plasma membrane of mammalian cells in tissue culture. Ultraviolet or two-photon lasers were used to induce small, localized wounds, and cell reactions were followed with time-lapse imaging. Propidium iodide (PI) entry in wounded cells was used to allow imaging of the plasma membrane opening and to quantify the rate of closure of single wounds. Mathematical fit of PI entry kinetics was used to estimate the diameter and the rate of closure of individual wounds. Characterization of PI fluorescence and diffusion gave us an estimation of wound sizes. Transfection of small interfering RNA or dominant-negative mutants of ESCRT subunits allowed us to assess their importance during plasma membrane repair. Last, using correlative-scanning electron microscopy we examined the ultrastructure of wounded plasma membranes. Results The various wounding methods used here revealed a systematic recruitment of ESCRTs to the plasma membrane. Wounding with a laser beam showed that ESCRTs—and in particular, ESCRT-III proteins—were specifically recruited to wound sites and were accumulated until wound closure. This recruitment depended on calcium, which is known to be a crucial signaling molecule for wound repair. The depletion of important ESCRT subunits such as CHMP4B, CHMP2A, or Vps4 was deleterious for a subpopulation of cells bearing small wounds (less than 100 nm in diameter). Correlative scanning electron microscopy and time-lapse imaging revealed that wounding was followed by ESCRT-positive membrane budding and shedding. Energy depletion did not prevent—and rather increased—ESCRT accumulation but prevented both membrane shedding and repair. Discussion These results show that ESCRT proteins play an important role in the detection and removal through the extracellular shedding of small wounds present at the plasma membrane. We propose that different mechanisms for membrane repair (patching, budding, or endocytosis) can be used by cells depending on the type and size of the wound. These mechanisms are stimulated by common early signaling events, such as calcium, but downstream events are likely to depend on the physiochemical characteristics of the wounds. ESCRT-positive plasma membrane shedding has been observed in a variety of normal and pathological conditions. It remains unclear whether these phenomena are linked to local plasma membrane damage and whether ESCRT-III proteins are involved in these processes. ESCRT Your Wound Away The ESCRT (endosomal sorting complex required for transport) protein complex plays a role in budding into multivesicular bodies, in cytokinesis, and in HIV budding. Now, Jimenez et al. (p. 10.1126/science.1247136, published online 30 January) propose a role for ESCRT proteins in wound repair at the plasma membrane. In vivo imaging, modeling, and electron microscopy were used to reveal how the ESCRTs participate in a rapid energy-independent, calcium-dependent, membrane-shedding process at the plasma membrane that reseals small wounds caused by toxins or laser treatment. ESCRT proteins repair small wounds in the plasma membrane by shearing off damaged portions. Plasma membrane damage can be triggered by numerous phenomena, and efficient repair is essential for cell survival. Endocytosis, membrane patching, or extracellular budding can be used for plasma membrane repair. We found that endosomal sorting complex required for transport (ESCRT), involved previously in membrane budding and fission, plays a critical role in plasma membrane repair. ESCRT proteins were recruited within seconds to plasma membrane wounds. Quantitative analysis of wound closure kinetics coupled to mathematical modeling suggested that ESCRTs are involved in the repair of small wounds. Real-time imaging and correlative scanning electron microscopy (SEM) identified extracellular buds and shedding at the site of ESCRT recruitment. Thus, the repair of certain wounds is ensured by ESCRT-mediated extracellular shedding of wounded portions.

Detection of GTP-Tubulin Conformation in Vivo Reveals a Role for GTP Remnants in Microtubule Rescues

Microtubules display dynamic instability, with alternating phases of growth and shrinkage separated by catastrophe and rescue events. The guanosine triphosphate (GTP) cap at the growing end of microtubules, whose presence is essential to prevent microtubule catastrophes in vitro, has been difficult to observe in vivo. We selected a recombinant antibody that specifically recognizes GTP-bound tubulin in microtubules and found that GTP-tubulin was indeed present at the plus end of growing microtubules. Unexpectedly, GTP-tubulin remnants were also present in older parts of microtubules, which suggests that GTP hydrolysis is sometimes incomplete during polymerization. Observations in living cells suggested that these GTP remnants may be responsible for the rescue events in which microtubules recover from catastrophe.

Interplay between microtubule dynamics and intracellular organization.

Microtubules are hollow tubes essential for many cellular functions such as cell polarization and migration, intracellular trafficking and cell division. They are polarized polymers composed of α and β tubulin that are, in most cells, nucleated at the centrosome at the center of the cell. Microtubule plus-ends are oriented towards the periphery of the cell and explore the cytoplasm in a very dynamic manner. Microtubule alternate between phases of growth and shrinkage in a manner described as dynamic instability. Their dynamics is highly regulated by multiple factors: tubulin post-translational modifications such as detyrosination or acetylation, and microtubule-associated proteins, among them the plus-tip tracking proteins. This regulation is necessary for microtubule functions in the cell. In this review, we will focus on the role of microtubules in intracellular organization. After an overview of the mechanisms responsible for the regulation of microtubule dynamics, the major roles of microtubules dynamics in organelle positioning and organization in interphase cells will be discussed. Conversely, the role of certain organelles, like the nucleus and the Golgi apparatus as microtubule organizing centers will be reviewed. We will then consider the role of microtubules in the establishment and maintenance of cell polarity using few examples of cell polarization: epithelial cells, neurons and migrating cells. In these cells, the microtubule network is reorganized and undergoes specific and local regulation events; microtubules also participate in the intracellular reorganization of different organelles to ensure proper cell differentiation.

Rab6A and Rab6A' GTPases play non-overlapping roles in membrane trafficking.

The closely related Rab6 isoforms, Rab6A and Rab6A′, have been shown to regulate vesicular trafficking within the Golgi and post‐Golgi compartments, but studies using dominant active or negative mutant suggested conflicting models. Here, we report that reduction in the expression of Rab6 isoform using specific small interfering RNA reveals noticeable differences in the Rab6A and Rab6A′ biological functions. Surprisingly, Rab6A seems to be largely dispensable in membrane trafficking events, whereas knocking down the expression of Rab6A′ hampers the intracellular transport of the retrograde cargo marker, the Shiga Toxin B‐subunit along the endocytic pathway, and causes defects in Golgi‐ associated protein recycling through the endoplasmic reticulum. We also showed that Rab6A′ is required for cell cycle progression through mitosis and identify Ile62 as a key residue for uncoupling Rab6A′ functions in mitosis and retrograde trafficking. Thus, our work shows that Rab6A and Rab6A′ perform different functions within the cell and suggests a novel role for Rab6A′ as the major Rab6 isoform regulating previously described Rab6‐dependent transport pathways.

Local palmitoylation cycles define activity-regulated postsynaptic subdomains

Local palmitoylation machinery has an instructive role in creating activity-responsive PSD-95 nanodomains, which contribute to postsynaptic density (re)organization.

Synchronization of secretory protein traffic in populations of cells

To dissect secretory traffic, we developed the retention using selective hooks (RUSH) system. RUSH is a two-state assay based on the reversible interaction of a hook protein fused to core streptavidin and stably anchored in the donor compartment with a reporter protein of interest fused to streptavidin-binding peptide (SBP). Biotin addition causes a synchronous release of the reporter from the hook. Using the RUSH system, we analyzed different transport characteristics of various Golgi and plasma membrane reporters at physiological temperature in living cells. Using dual-color simultaneous live-cell imaging of two cargos, we observed intra- and post-Golgi segregation of cargo traffic, consistent with observation in other systems. We show preliminarily that the RUSH system is usable for automated screening. The system should help increase the understanding of the mechanisms of trafficking and enable screens for molecules that perturb pathological protein transport.

A plus-end raft to control microtubule dynamics and function

Cells require a properly oriented and organised microtubule array to transmit positional information. Recent data have revealed a heterogeneous population of microtubule-binding proteins that accumulates mainly at distal ends of polymerising microtubules. Two mechanisms may account for this concentration: transient immobilisation, which involves association of proteins with growing ends, followed by release more proximally; and deposition at ends via a molecular motor. As with lipid rafts, protein concentration at distal ends may allow a cascade of interactions in the restricted area of a microtubule plus end. This may, in turn, control the dynamic behaviour of this cytoskeletal network and its anchoring to other structures.

Preferential binding of a kinesin-1 motor to GTP-tubulin–rich microtubules underlies polarized vesicle transport

The high affinity of KIF5 for microtubules rich in GTP-tubulin results in polarized motor protein accumulation at axonal tips in neurons and may underlie polarized vesicle transport.