Protein localization by actin treadmilling and molecular motors regulates stereocilia shape and treadmilling rate

We present a physical model that describes the active localization of actin-regulating proteins inside stereocilia during steady-state conditions. The mechanism of localization is through the interplay of free diffusion and directed motion, which is driven by coupling to the treadmilling actin filaments and to myosin motors that move along the actin filaments. The resulting localization of both the molecular motors and their cargo is calculated, and is found to have an exponential (or steeper) profile. This localization can be at the base (driven by actin retrograde flow and minus-end myosin motors), or at the stereocilia tip (driven by plus-end myosin motors). The localization of proteins that influence the actin depolymerization and polymerization rates allow us to describe the narrow shape of the stereocilia base, and the observed increase of the actin polymerization rate with the stereocilia height.     

Rate of treadmilling of actin filaments in vitro

Actin filaments capped at the barbed ends were formed by polymerizing monomeric actin onto a gelsolin-actin complex. The rate of depolymerization and polymerization of the pointed ends was determined by diluting gelsolin-capped actin filaments into various concentrations of monomeric actin. Under the conditions of the experiments (100 mM-KCl, 2 mM-MgCl2 at 37 degrees C) the rate constant of dissociation of subunits both from a shortening and a lengthening filament was found to be 0.21 s-1. As the rate of dissociation of subunits from the slow pointed end determines the rate of treadmilling, it is concluded that actin filaments treadmill with a rate of about 2 micron/h.     

Implications of treadmilling for the stability and polarity of actin and tubulin polymers in vivo

In this report, we examine how the cell can selectively stabilize anchored filaments and suppress spontaneous filament assembly. Because microtubules and actin filaments have an organized distribution in cells, the cell must have a mechanism for suppressing spontaneous and random polymerization. Though the mechanism for suppressing spontaneous polymerization is unknown, an unusual property of these filaments has been demonstrated recently, i.e., under steady-stae conditions, in vitro actin filaments and microtubules can exhibit a flux of subunits through the polymers called "treadmilling." In vivo, however, most, if not all, of these polymers are attached at one end to specific structures and treadmilling should not occur. The function of treadmilling in vivo is, therefore, unclear at present. However, as shown here, the same physicochemical property of coupling assembly to ATP or GTP hydrolysis that leads to treadmilling in vitro can act to selectively stabilize anchored polymers in vivo. I show here that the theory of treadmilling implies that the concentration of subunits necessary for assembly of the nonanchored polymer will in general be higher than the concentration necessary for the assembly of polymers anchored with a specific polarity. This disparity in the monomer concentrations required for assembly can lead to a selective stabilization of anchored polymers and complete suppression of spontaneous polymerization at apparent equilibrium in vivo. It is possible, therefore, that the phenomenon of treadmilling is an in vitro manifestation of a mechanism designed to use ATP or GTP hydrolysis to control the spatial organization of filaments in the cell.     

Chemokine triggered integrin activation and actin remodeling events guiding lymphocyte migration across vascular barriers

Chemokine signals activate leukocyte integrins and actin remodeling machineries critical for leukocyte adhesion and motility across vascular barriers. The arrest of leukocytes at target blood vessel sites depends on rapid conformational activation of their α4 and β2 integrins by the binding of endothelial-displayed chemokines to leukocyte Gi-protein coupled receptors (GPCRs). A universal regulator of this event is the integrin-actin adaptor, talin1. Chemokine-stimulated GPCRs can transmit within fractions of seconds signals via multiple Rho GTPases, which locally raise plasma membrane levels of the talin activating phosphatidyl inositol, PtdIns(4,5)P2 (PIP2). Additional pools of GPCR stimulated Rac-1 and Rap-1 GTPases together with GPCR stimulated PLC and PI3K family members regulate the turnover of focal contacts of leukocyte integrins, induce the collapse of leukocyte microvilli, and promote polarized leukocyte crawling in search of exit cues. Concomitantly, other leukocyte GTPases trigger invasive protrusions into and between endothelial cells in search of basolateral chemokine exit cues. We will review here major findings and open questions related to these sequential guiding activities of endothelial presented chemokines, focusing mainly on lymphocyte-endothelial interactions as a paradigm for other leukocytes.     

Exchange of actin subunits at the leading edge of living fibroblasts: possible role of treadmilling

Previous observations indicated that the lamellipodium ("leading edge") of fibroblasts contains a dense meshwork, as well as numerous bundles (microspikes) of actin filaments. Most, if not all, of the filaments have a uniform polarity, with the "barbed" end associated with the membrane. I investigated whether and how actin subunits exchange in this region by microinjecting living gerbil fibroma cells (IMR-33) with actin that had been labeled with iodoacetamidotetramethylrhodamine. After incorporation of the labeled actin into the lamellipodium, I used a laser microbeam to photobleach a 3-4-micron region at and surrounding a microspike, without disrupting the integrity of the structure. I then recorded the pattern of fluorescence recovery and analyzed it using a combination of TV image intensification and digital image processing techniques. Fluorescence recovery was first detected near the edge of the cell and then moved toward the cell's center at a constant rate of 0.79 +/- 0.31 micron/min. When only part of the lamellipodium near the edge of the cell was photobleached, the bleached spot also moved toward the cell's center and through an area unbleached by the laser beam. These results indicated that steady state incorporation of actin subunits occurred predominantly at the membrane-associated end of actin filaments, and that actin subunits in the lamellipodium underwent a constant movement toward the center of the cell. I suggest that treadmilling, possibly in combination with other molecular interactions, may provide an effective mechanism for the movement of actin subunits and the protrusion of cytoplasm in the lamellipodium of fibroblasts.     

Arp2/3 complex and actin depolymerizing factor/cofilin in dendritic organization and treadmilling of actin filament array in lamellipodia.

The leading edge (approximately 1 microgram) of lamellipodia in Xenopus laevis keratocytes and fibroblasts was shown to have an extensively branched organization of actin filaments, which we term the dendritic brush. Pointed ends of individual filaments were located at Y-junctions, where the Arp2/3 complex was also localized, suggesting a role of the Arp2/3 complex in branch formation. Differential depolymerization experiments suggested that the Arp2/3 complex also provided protection of pointed ends from depolymerization. Actin depolymerizing factor (ADF)/cofilin was excluded from the distal 0.4 micrometer++ of the lamellipodial network of keratocytes and in fibroblasts it was located within the depolymerization-resistant zone. These results suggest that ADF/cofilin, per se, is not sufficient for actin brush depolymerization and a regulatory step is required. Our evidence supports a dendritic nucleation model (Mullins, R.D., J.A. Heuser, and T.D. Pollard. 1998. Proc. Natl. Acad. Sci. USA. 95:6181-6186) for lamellipodial protrusion, which involves treadmilling of a branched actin array instead of treadmilling of individual filaments. In this model, Arp2/3 complex and ADF/cofilin have antagonistic activities. Arp2/3 complex is responsible for integration of nascent actin filaments into the actin network at the cell front and stabilizing pointed ends from depolymerization, while ADF/cofilin promotes filament disassembly at the rear of the brush, presumably by pointed end depolymerization after dissociation of the Arp2/3 complex.     

Fluorescent dyes for lymphocyte migration and proliferation studies

Fluorescent dyes are increasingly being exploited to track lymphocyte migration and proliferation. The present paper reviews the properties and performance of some 14 different fluorescent dyes that have been used during the last 20 years to monitor lymphocyte migration. Of the 14 dyes discussed, two stand out as being the most versatile in terms of long-term tracking of lymphocytes and their ability to quantify lymphocyte proliferation. They are the intracellular covalent coupling dye carboxyfluorescein diacetate succinimidyl ester (CFSE) and the membrane inserting dye PKH26. Both dyes have the advantage that they can be used to track cell division, both in vitro and in vivo, due to the progressive halving of the fluorescence intensity of the dyes in cells after each division. However, CFSE appears to have the edge over PKH26 based on homogeneity of lymphocyte staining and cost. Two other fluorescent dyes, although not suitable for lymphocyte proliferation studies, are valuable tracking dyes for short-term (up to 3 day) lymphocyte migration experiments, namely the DNA-binding dye Hoechst 33342 and the cytoplasmic dye calcein. In the future it is highly likely that additional fluorescent dyes, with different spectral properties to CFSE, will become available, as well as membrane inserting fluorescent dyes that more homogeneously label lymphocytes than PKH26.     

Calcium signalling in growth cone migration

Growth cones, the motile structures at the tips of advancing axons and dendrites, respond to a wide range of cues by either turning towards or away from the cue. Cytosolic calcium signals appear to mediate a large fraction of both types of response. Calcium signals can be generated by influx through plasma membrane channels or by release from intracellular stores. While neurotransmitters can elicit calcium influx through ionotropic receptors, other chemical cues open plasma membrane voltage gated calcium channels by a mechanism other than a change of membrane voltage. In general attractive cues generate spatially and temporally restricted calcium increases that are difficult to detect using conventional indicators. One target for these calcium signals is calmodulin dependent protein kinase II. Repulsive cues generate spatially and temporally more diffuse calcium increases that can be more readily detected using fluorescent indicators. One target for these is the phosphatase calcineurin, which may act by dephosphorylating GAP43 and allowing the latter to cap actin filaments.     

Integrin signalling in directed cell migration

Migrating cells tend to continue moving in the same direction, a property called persistence. During migration, cells, by definition, form new adhesions at their front and break old adhesions at the rear. We hypothesize that the distinction between new adhesions at the front and older adhesions at the rear plays a major role in directional persistence. We propose specific mechanisms of persistence on the basis of known properties of integrin signals, in hope of stimulating investigation of these ideas.     

Adhesion molecules controlling lymphocyte migration

Two newly characterized structural families of adhesion molecules, in concert with known members of the integrin and immunoglobulin supergene families, mediate the interaction of circulating lymphoid cells with the vessel wall. The Hermes/CD44 antigen family participates in attachment to multiple vascular beds and consists of a common polypeptide core showing amino-terminal homology to cartilage link proteins. In contrast, the node-specific homing receptor Mel-14 consists of substructures homologous to calcium-dependent lectins, EGF, and complement binding proteins. The sequence of Mel-14 provides structural support for the hypothesis that lectin-carbohydrate interactions mediate physiologically significant adhesion events in the course of lymphocyte recirculation. The discovery of a similar structure in ELAM-1 and GMP-140 extends the reach of this family to other leukocyte and platelet interactions with the vessel wall.     

The F-BAR protein Hof1 tunes formin activity to sculpt actin cables during polarized growth

Asymmetric cell growth and division rely on polarized actin cytoskeleton remodeling events, the regulation of which is poorly understood. In budding yeast, formins stimulate the assembly of an organized network of actin cables that direct polarized secretion. Here we show that the Fer/Cip4 homology–Bin amphiphysin Rvs protein Hof1, which has known roles in cytokinesis, also functions during polarized growth by directly controlling the activities of the formin Bnr1. A mutant lacking the C-terminal half of Hof1 displays misoriented and architecturally altered cables, along with impaired secretory vesicle traffic. In vitro, Hof1 inhibits the actin nucleation and elongation activities of Bnr1 without displacing the formin from filament ends. These effects depend on the Src homology 3 domain of Hof1, the formin homology 1 (FH1) domain of Bnr1, and Hof1 dimerization, suggesting a mechanism by which Hof1 “restrains” the otherwise flexible FH1-FH2 apparatus. In vivo, loss of inhibition does not alter actin levels in cables but, instead, cable shape and functionality. Thus Hof1 tunes formins to sculpt the actin cable network.     

Requirements for T lymphocyte migration in explanted lymph nodes

Although the requirements for T lymphocyte homing to lymph nodes (LNs) are well studied, much less is known about the requirements for T lymphocyte locomotion within LNs. Imaging of murine T lymphocyte migration in explanted LNs using two-photon laser-scanning fluorescence microscopy provides an opportunity to systematically study these requirements. We have developed a closed system for imaging an intact LN with controlled temperature, oxygenation, and perfusion rate. Naive T lymphocyte locomotion in the deep paracortex of the LN required a perfusion rate of >13 microm/s and a partial pressure of O(2) (pO(2)) of >7.4%. Naive T lymphocyte locomotion in the subcapsular region was 38% slower and had higher turning angles and arrest coefficients than naive T lymphocytes in the deep paracortex. T lymphocyte activation decreased the requirement for pO(2), but also decreased the speed of locomotion in the deep paracortex. Although CCR7(-/-) naive T cells displayed a small reduction in locomotion, systemic treatment with pertussis toxin reduced naive T lymphocyte speed by 59%, indicating a contribution of Galpha(i)-mediated signaling, but involvement of other G protein-coupled receptors besides CCR7. Receptor knockouts or pharmacological inhibition in the adenosine, PG/lipoxygenase, lysophosphatidylcholine, and sphingosine-1-phosphate pathways did not individually alter naive T cell migration. These data implicate pO(2), tissue architecture, and G-protein coupled receptor signaling in regulation of naive T lymphocyte migration in explanted LNs.     

Abnormal migration of T lymphocyte clones

Several in vitro T cell clones were markedly deficient in their ability to home to peripheral lymphoid tissue. This was found for an alloreactive noncytolytic clone, a soluble antigen- (KLH)specific line, and cytotoxic clones specific for allogeneic cells and for Abelson virus-induced lymphoma cells. This abnormal circulation pattern was probably caused by the lack of the receptors of the lymphocytes for high endothelial venules (HEV), as implied by the lack of binding of these T cells to HEV in frozen sections of mouse lymph node and Peyer's patches. The loss of surface receptors that are necessary for normal lymphocyte migration may thereby alter the in vivo function of adoptively transferred T cells.     

Cofilin tunes the nucleotide state of actin filaments and severs at bare and decorated segment boundaries

Actin-based motility demands the spatial and temporal coordination of numerous regulatory actin-binding proteins (ABPs), many of which bind with affinities that depend on the nucleotide state of actin filament. Cofilin, one of three ABPs that precisely choreograph actin assembly and organization into comet tails that drive motility in vitro, binds and stochastically severs aged ADP actin filament segments of de novo growing actin filaments. Deficiencies in methodologies to track in real time the nucleotide state of actin filaments, as well as cofilin severing, limit the molecular understanding of coupling between actin filament chemical and mechanical states and severing. We engineered a fluorescently labeled cofilin that retains actin filament binding and severing activities. Because cofilin binding depends strongly on the actin-bound nucleotide, direct visualization of fluorescent cofilin binding serves as a marker of the actin filament nucleotide state during assembly. Bound cofilin allosterically accelerates P(i) release from unoccupied filament subunits, which shortens the filament ATP/ADP-P(i) cap length by nearly an order of magnitude. Real-time visualization of filament severing indicates that fragmentation scales with and occurs preferentially at boundaries between bare and cofilin-decorated filament segments, thereby controlling the overall filament length, depending on cofilin binding density.     

Transplantation stimulates interstitial cell migration in hydra

Migration of interstitial cells and nerve cell precursors was analyzed in Hydra magnipapillata and Hydra vulgaris (formerly Hydra attenuata). Axial grafts were made between [3H]thymidine-labeled donor and unlabeled host tissue. Migration of labeled cells into the unlabeled half was followed for 4 days. The results indicate that the rate of migration was initially high and then slowed on Days 2-4. Regrafting fresh donor tissue on Days 2-4 maintained high levels of migration. Thus, migration appears to be stimulated by the grafting procedure itself.