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Actin polymerizes near the leading edge of nerve growth cones, and actin filaments show retrograde movement in filopodia and lamellipodia. Linkage between actin filament retrograde flow and cell adhesion molecules (CAMs) in growth cones is thought to be one of the mechanisms for axon outgrowth and guidance. However, the molecular basis for this linkage remains elusive. Here, we show that shootin1 interacts with both actin filament retrograde flow and L1-CAM in axonal growth cones of cultured rat hippocampal neurons, thereby mediating the linkage between them. Impairing this linkage, either by shootin1 RNA interference or disturbing the interaction between shootin1 and actin filament flow, inhibited L1-dependent axon outgrowth, whereas enhancing the linkage by shootin1 overexpression promoted neurite outgrowth. These results strengthen the actin flow-CAM linkage model ('clutch' model) for axon outgrowth and suggest that shootin1 is a key molecule involved in this mechanism.

Axonal growth cones migrate along the correct paths during development, not only directed by guidance cues but also contacted by local environment via cell adhesion molecules (CAMs). Asymmetric [Ca.sup.2+] elevations in the growth cone cytosol induce both attractive and repulsive turning in response to the guidance cues (Zheng, J.Q. 2000. Nature. 403:89-93; Henley, J.R., K.H. Huang, D. Wang, and M.M. Poo. 2004. Neuron. 44:909-916). Here, we show that CAMs regulate the activity of ryanodine receptor type 3 (RyR3) via cAMP and protein kinase A in dorsal root ganglion neurons. The activated RyR3 mediates [Ca.sup.2+]-induced [Ca.sup.2+] release (CICR) into the cytosol, leading to attractive turning of the growth cone. In contrast, the growth cone exhibits repulsion when [Ca.sup.2+] signals are not accompanied by RyR3-mediated CICR. We also propose that the source of [Ca.sup.2+] influx, rather than its amplitude or the baseline [Ca.sup.2-] level, is the primary determinant of the turning direction. In this way, axon-guiding and CAM-derived signals are integrated by RyR3, which serves as a key regulator of growth cone navigation.

The cell adhesion molecule L1 (L1-CAM) plays critical roles in neurite growth. Its cytoplasmic domain (L1CD) binds to ankyrins that associate with the spectrin-actin network. This paper demonstrates that L1-CAM interactions with [ankyrin.sub.B] (but not with [ankyrin.sub.G]) are involved in the initial formation of neurites. In the membranous protrusions surrounding the soma before neuritogenesis, filamentous actin (F-actin) and [ankyrin.sub.G] continuously move toward the soma (retrograde flow). Bead-tracking experiments show that [ankyrin.sub.B] mediates L1-CAM coupling with retrograde F-actin flow in these perisomatic structures. Ligation of the L1-CAM ectodomain by an immobile substrate induces L1CD-[ankyrin.sub.B] binding and the formation of stationary [ankyrin.sub.B] clusters. Neurite initiation preferentially occurs at the site of these clusters. In contrast, [ankyrin.sub.B] is involved neither in L1-CAM coupling with F-actin flow in growth cones nor in L1-based neurite elongation. Our results indicate that [ankyrin.sub.B] promotes neurite initiation by acting as a component of the clutch module that transmits traction force generated by F-actin flow to the extracellular substrate via L1-CAM.

Lyso‐phosphatidyl β‐D‐glucoside (lysoPtdGlc) is during embryonic development a neurite growth cone guidance cue associated with G‐protein coupled receptor 55, and has been found to be involved in a variety of physiological and pathological events, including inflammatory pain, and cancer. Hence, the ability to prepare large quantities of lysoPtdGlc is an important objective. Herein, we report the convergent chemical synthesis of lysoPtdGlc and its glyceryl epimer (S‐lysoPtdGlc). High stereocontrol during β‐glycosyl H‐phosphonate construction was achieved using a microfluidic approach, and common but undesirable regioisomerization due to acyl migration on the glyceryl subunit was successfully suppressed during deprotection. Purification of the final product was optimized to obtain sufficiently large amounts of lysoPtdGlc and S‐lysoPtdGlc for biological assaying purposes. [ABSTRACT FROM AUTHOR]

Skin development is tightly temporally coordinated with its sensory innervation, which consists of the peripheral branches of the dorsal root ganglion (DRG) axons. Various studies suggest that the skin produces a long‐range attractant for the sensory axons. However, the exact identity of the guidance cue(s) remains unclear. To reveal the detailed molecular mechanism that controls DRG axon guidance and targeting, manipulation of specific skin layers at specific time points are required. To test a variety of attractants that can be expressed in specific skin layers at specific timepoints, we combined in utero electroporation with the Tol2 transposon system to induce long‐term transgene expression in the developing mouse skin, including in the highly proliferative epidermal stem cells (basal layer) and their descendants (spinous and granular layer cells). The plasmid solution was injected as close to the hindpaw plantar surface as possible. Immediately, electric pulses were passed through the embryo to transduce the plasmid DNA into hindpaw skin cells. Balancing outcome measurements including: embryo survival, transfection efficiency, and the efficiency of transgene integration into host cells, we found that IUE was best performed on E13.5, and using an electroporation voltage of 34V. After immunostaining embryonic and early postnatal skin tissue sections for keratinocyte and sensory axon markers, we observe the growth of axons into skin epidermal layers including areas expressing EGFP. Therefore, this method is useful for studying the interaction between axon growth and epidermal cell division/differentiation. [ABSTRACT FROM AUTHOR]

Lysophosphatidyl glucoside (LPGlc) is a structurally unique glycolipid that acts as a guidance cue for extending axons during central nervous system development by activating the class A G protein coupled receptor (GPR) 55 of spinal cord sensory axons. GPR55 not only plays an important role during development, but is also implicated in many disease states, rendering molecules that target GPR55 of widespread interest. In this study, we developed synthetic access to a novel class of LPGlc analogues featuring a squaryl diamide group as surrogate for the phosphodiester. We report the facile synthesis of a series of LPGlc analogues, their GPR dependent biological activity and a systematic analysis of the structure–activity relationship in regards to GPR55 modulation. The lead compound featuring identical configuration at all stereocenters compared to natural LPGlc exhibits an activity to repel axons of dorsal root ganglion (DGR) nociceptive neurons. [ABSTRACT FROM AUTHOR]