Growth and site-specific organization of micron-scale biomolecular devices on living mammalian cells.

Mesoscale molecular assemblies on the cell surface, such as cilia and filopodia, integrate information, control transport and amplify signals. Designer cell-surface assemblies could control these cellular functions. Such assemblies could be constructed from synthetic components ex vivo, making it possible to form such structures using modern nanoscale self-assembly and fabrication techniques, and then oriented on the cell surface. Here we integrate synthetic devices, micron-scale DNA nanotubes, with mammalian cells by anchoring them by their ends to specific cell surface receptors. These filaments can measure shear stresses between 0-2 dyn/cm2, a regime important for cell signaling. Nanotubes can also grow while anchored to cells, thus acting as dynamic cell components. This approach to cell surface engineering, in which synthetic biomolecular assemblies are organized with existing cellular architecture, could make it possible to build new types of sensors, machines and scaffolds that can interface with, control and measure properties of cells.

A distinct parabrachial-to-lateral hypothalamus circuit for motivational suppression of feeding by nociception.

The motivation to eat is not only shaped by nutrition but also competed by external stimuli including pain. How the mouse hypothalamus, the feeding regulation center, integrates nociceptive inputs to modulate feeding is unclear. Within the key nociception relay center parabrachial nucleus (PBN), we demonstrated that neurons projecting to the lateral hypothalamus (LHPBN) are nociceptive yet distinct from danger-encoding central amygdala-projecting (CeAPBN) neurons. Activation of LHPBN strongly suppressed feeding by limiting eating frequency and also reduced motivation to work for food reward. Refined approach-avoidance paradigm revealed that suppression of LHPBN, but not CeAPBN, sustained motivation to obtain food. The effect of LHPBN neurons on feeding was reversed by suppressing downstream LHVGluT2 neurons. Thus, distinct from a circuit for fear and escape responses, LHPBN neurons channel nociceptive signals to LHVGluT2 neurons to suppress motivational drive for feeding. Our study provides a new perspective in understanding feeding regulation by external competing stimuli.

Autonomy declared by primary cilia through compartmentalization of membrane phosphoinositides.

The primary cilium is a cell surface projection from plasma membrane which transduces external stimuli to diverse signaling pathways. To function as an independent signaling organelle, the molecular composition of the ciliary membrane has to be distinct from that of the plasma membrane. Here, we review recent findings which have deepened our understanding of the unique yet dynamic phosphoinositide profile found in the primary cilia.

Dynamic Remodeling of Membrane Composition Drives Cell Cycle through Primary Cilia Excision.

The life cycle of a primary cilium begins in quiescence and ends prior to mitosis. In quiescent cells, the primary cilium insulates itself from contiguous dynamic membrane processes on the cell surface to function as a stable signaling apparatus. Here, we demonstrate that basal restriction of ciliary structure dynamics is established by the cilia-enriched phosphoinositide 5-phosphatase, Inpp5e. Growth induction displaces ciliary Inpp5e and accumulates phosphatidylinositol 4,5-bisphosphate in distal cilia. This change triggers otherwise-forbidden actin polymerization in primary cilia, which excises cilia tips in a process we call cilia decapitation. While cilia disassembly is traditionally thought to occur solely through resorption, we show that an acute loss of IFT-B through cilia decapitation precedes resorption. Finally, we propose that cilia decapitation induces mitogenic signaling and constitutes a molecular link between the cilia life cycle and cell-division cycle. This newly defined ciliary mechanism may find significance in cell proliferation control during normal development and cancer.

An intelligent nano-antenna: Primary cilium harnesses TRP channels to decode polymodal stimuli.

The primary cilium is a solitary hair-like organelle on the cell surface that serves as an antenna sensing ever-changing environmental conditions. In this review, we will first recapitulate the molecular basis of the polymodal sensory function of the primary cilia, specifically focusing on transient receptor potential (TRP) channels that accumulate inside the organelle and conduct calcium ions (Ca(2+)). Each subfamily member, namely TRPP2 TRPP3, TRPC1 and TRPV4, is gated by multiple environmental factors, including chemical (receptor ligands, intracellular second messengers such as Ca(2+)), mechanical (fluid shear stress, hypo-osmotic swelling), or physical (temperature, voltage) stimuli. Both activity and heterodimer compositions of the TRP channels may be dynamically regulated for precise tuning to the varying dynamic ranges of the individual input stimuli. We will thus discuss the potential regulation of TRP channels by local second messengers. Despite its reported importance in embryonic patterning and tissue morphogenesis, the precise functional significance of the downstream Ca(2+) signals of the TRP channels remains unknown. We will close our review by featuring recent technological advances in visualizing and analyzing signal transduction inside the primary cilia, together with current perspectives illuminating the functional significance of intraciliary Ca(2+) signals.

Genetically encoded calcium indicator illuminates calcium dynamics in primary cilia.

Visualization of signal transduction in live primary cilia constitutes a technical challenge owing to the organelle's submicrometer dimensions and close proximity to the cell body. Using a genetically encoded calcium indicator targeted to primary cilia, we visualized calcium signaling in cilia of mouse fibroblasts and kidney cells upon chemical or mechanical stimulation with high specificity, high sensitivity and wide dynamic range.

Visualizing molecular diffusion through passive permeability barriers in cells: conventional and novel approaches.

Diffusion barriers are universal solutions for cells to achieve distinct organizations, compositions, and activities within a limited space. The influence of diffusion barriers on the spatiotemporal dynamics of signaling molecules often determines cellular physiology and functions. Over the years, the passive permeability barriers in various subcellular locales have been characterized using elaborate analytical techniques. In this review, we will summarize the current state of knowledge on the various passive permeability barriers present in mammalian cells. We will conclude with a description of several conventional techniques and one new approach based on chemically inducible diffusion trap (CIDT) for probing permeable barriers.

Chemically inducible diffusion trap at cilia reveals molecular sieve-like barrier.

Primary cilia function as specialized compartments for signal transduction. The stereotyped structure and signaling function of cilia inextricably depend on the selective segregation of molecules in cilia. However, the fundamental principles governing the access of soluble proteins to primary cilia remain unresolved. We developed a methodology termed 'chemically inducible diffusion trap at cilia' to visualize the diffusion process of a series of fluorescent proteins ranging in size from 3.2 nm to 7.9 nm into primary cilia. We found that the interior of the cilium was accessible to proteins as large as 7.9 nm. The kinetics of ciliary accumulation of this panel of proteins was exponentially limited by their Stokes radii. Quantitative modeling suggests that the diffusion barrier operates as a molecular sieve at the base of cilia. Our study presents a set of powerful, generally applicable tools for the quantitative monitoring of ciliary protein diffusion under both physiological and pathological conditions.

Rapidly relocating molecules between organelles to manipulate small GTPase activity.

Chemically inducible rapid manipulation of small GTPase activity has proven a powerful approach to dissect complex spatiotemporal signaling of these molecular switches. However, overexpression of these synthetic molecular probes freely in the cytosol often results in elevated background activity before chemical induction, which perturbs the cellular basal state and thereby limits their wide application. As a fundamental solution, we have rationally designed and newly developed a strategy to remove unwanted background activity without compromising the extent of induced activation. By exploiting interaction between a membrane lipid and its binding protein, target proteins were translocated from one organelle to another on a time scale of seconds. This improved strategy now allows for rapid manipulation of small GTPases under a physiological state, thus enabling fine dissection of sophisticated signaling processes shaped by these molecules.

Informatics-based analysis of mechanosignaling in the laminopathies.

The A- and B-type lamins are the primary building blocks of the lamina-a proteinaceous meshwork underlying the nuclear envelope (NE). In the last decade, some 25 diseases have been linked to mutations in genes encoding proteins of the NE and lamina, with about half being caused by mutations in the Lamin A gene. Cells, either from patients or from mice carrying lamin mutations, frequently exhibit deformed nuclei accompanied by compromised mechanical properties in both the nucleus and the cytoplasm, implying that defects in the mechanical integrity of the nuclei and in mechanosignaling contribute to the pathology of these diseases. We describe a procedure to study total gene expression of mutant cells subjected to uniaxial mechanical strain by culturing them on a deformable surface. Using our procedure, enough high-quality RNA can be collected from these samples for microarray and informatics analysis. Such analysis may provide valuable information regarding the changes in gene expression and signaling pathways that may underlie the pathologies of the various diseases, which in turn may arise as a consequence of defective responses to mechanical strain.