Detection of rare antigen-presenting cells through T cell-intrinsic meandering motility, mediated by Myo1g.

To mount an immune response, T lymphocytes must successfully search for foreign material bound to the surface of antigen-presenting cells. How T cells optimize their chances of encountering and responding to these antigens is unknown. T cell motility in tissues resembles a random or Levy walk and is regulated in part by external factors including chemokines and lymph-node topology, but motility parameters such as speed and propensity to turn may also be cell intrinsic. Here we found that the unconventional myosin 1g (Myo1g) motor generates membrane tension, enforces cell-intrinsic meandering search, and enhances T-DC interactions during lymph-node surveillance. Increased turning and meandering motility, as opposed to ballistic motility, is enhanced by Myo1g. Myo1g acts as a "turning motor" and generates a form of cellular "flânerie." Modeling and antigen challenges show that these intrinsically programmed elements of motility search are critical for the detection of rare cognate antigen-presenting cells.

Enterocyte microvillus-derived vesicles detoxify bacterial products and regulate epithelial-microbial interactions.

The continuous monolayer of intestinal epithelial cells (IECs) lining the gut lumen functions as the site of nutrient absorption and as a physical barrier to prevent the translocation of microbes and associated toxic compounds into the peripheral vasculature. IECs also express host defense proteins such as intestinal alkaline phosphatase (IAP), which detoxify bacterial products and prevent intestinal inflammation. Our laboratory recently showed that IAP is enriched on vesicles that are released from the tips of IEC microvilli and accumulate in the intestinal lumen. Here, we show that these native "lumenal vesicles" (LVs) (1) contain catalytically active IAP that can dephosphorylate lipopolysaccharide (LPS), (2) cluster on the surface of native lumenal bacteria, (3) prevent the adherence of enteropathogenic E. coli (EPEC) to epithelial monolayers, and (4) limit bacterial population growth. We also find that IECs upregulate LV production in response to EPEC and other Gram-negative pathogens. Together, these results suggest that microvillar vesicle shedding represents a novel mechanism for distributing host defense machinery into the intestinal lumen and that microvillus-derived LVs modulate epithelial-microbial interactions.

Myosin-1a: A motor for microvillar membrane movement and mechanics.

Myosin-1a is one of eight monomeric, membrane binding class I myosins expressed in vertebrates.1 As the most abundant actin-based motor protein found in the enterocyte microvillus, myosin-1a has long been known to interact with the apical membrane via a highly basic C-terminal tail domain.2 Several recent studies shed light on possible functional consequences of this protein/lipid interaction. In vitro and in vivo studies of microvillar function have revealed that myosin-1a can move apical membrane along core actin bundles, leading to the release of small vesicles from microvillar tips.3,4 Additional studies indicate that myosin-1a and other class I myosins contribute to membrane-cytoskeleton adhesion, which enables the apical membrane to resist deformation.5 These findings clearly position myosin-1a as an important player in apical membrane movement and structural stability. How this motor is able to fulfill these two seemingly distinct functions is currently unclear, but will serve as the focus of our discussion below.

Myosin motor function: the ins and outs of actin-based membrane protrusions.

Cells build plasma membrane protrusions supported by parallel bundles of F-actin to enable a wide variety of biological functions, ranging from motility to host defense. Filopodia, microvilli and stereocilia are three such protrusions that have been the focus of intense biological and biophysical investigation in recent years. While it is evident that actin dynamics play a significant role in the formation of these organelles, members of the myosin superfamily have also been implicated as key players in the maintenance of protrusion architecture and function. Based on a simple analysis of the physical forces that control protrusion formation and morphology, as well as our review of available data, we propose that myosins play two general roles within these structures: (1) as cargo transporters to move critical regulatory components toward distal tips and (2) as mediators of membrane-cytoskeleton adhesion.

Differential localization and dynamics of class I myosins in the enterocyte microvillus.

Epithelial cells lining the intestinal tract build an apical array of microvilli known as the brush border. Each microvillus is a cylindrical membrane protrusion that is linked to a supporting actin bundle by myosin-1a (Myo1a). Mice lacking Myo1a demonstrate no overt physiological symptoms, suggesting that other myosins may compensate for the loss of Myo1a in these animals. To investigate changes in the microvillar myosin population that may limit the Myo1a KO phenotype, we performed proteomic analysis on WT and Myo1a KO brush borders. These studies revealed that WT brush borders also contain the short-tailed class I myosin, myosin-1d (Myo1d). Myo1d localizes to the terminal web and striking puncta at the tips of microvilli. In the absence of Myo1a, Myo1d peptide counts increase twofold; this motor also redistributes along the length of microvilli, into compartments normally occupied by Myo1a. FRAP studies demonstrate that Myo1a is less dynamic than Myo1d, providing a mechanistic explanation for the observed differential localization. These data suggest that Myo1d may be the primary compensating class I myosin in the Myo1a KO model; they also suggest that dynamics govern the localization and function of different yet closely related myosins that target common actin structures.

Control of cell membrane tension by myosin-I.

All cell functions that involve membrane deformation or a change in cell shape (e.g., endocytosis, exocytosis, cell motility, and cytokinesis) are regulated by membrane tension. While molecular contacts between the plasma membrane and the underlying actin cytoskeleton are known to make significant contributions to membrane tension, little is known about the molecules that mediate these interactions. We used an optical trap to directly probe the molecular determinants of membrane tension in isolated organelles and in living cells. Here, we show that class I myosins, a family of membrane-binding, actin-based motor proteins, mediate membrane/cytoskeleton adhesion and thus, make major contributions to membrane tension. These studies show that class I myosins directly control the mechanical properties of the cell membrane; they also position these motor proteins as master regulators of cellular events involving membrane deformation.

All-optical constant-force laser tweezers.

Optical tweezers are a powerful tool for the study of single biomolecules. Many applications require that a molecule be held under constant tension while its extension is measured. We present two schemes based on scanning-line optical tweezers to accomplish this, providing all-optical alternatives to force-clamp traps that rely on electronic feedback to maintain constant-force conditions for the molecule. In these schemes, a laser beam is rapidly scanned along a line in the focal plane of the microscope objective, effectively creating an extended one-dimensional optical potential over distances of up to 8 microm. A position-independent lateral force acting on a trapped particle is created by either modulating the laser beam intensity during the scan or by using an asymmetric beam profile in the back focal plane of the microscope objective. With these techniques, forces of up to 2.69 pN have been applied over distances of up to 3.4 microm with residual spring constants of <26.6 fN/microm. We used these techniques in conjunction with a fast position measurement scheme to study the relaxation of lambda-DNA molecules against a constant external force with submillisecond time resolution. We compare the results to predictions from the wormlike chain model.

Fast position measurements with scanning line optical tweezers.

Scanning line optical tweezers are a powerful tool for the study of colloidal or biomolecular systems in the low-force regime. We present a fast, high-resolution particle position measurement scheme that extends the capabilities of these instruments into the realm of dynamic measurements. The technique is based on synchronous detection of forward-scattered laser light during a line scan. We demonstrate a position resolution of better than 50 nm for bandwidths of as much as 40 kHz for pairs of microspheres trapped in a flat line potential at center-to-center separations of 1.7-6 microm.