Lihsia Chen

Ankyrins and cellular targeting of diverse membrane proteins to physiological sites

Ankyrins are spectrin-binding proteins that associate via ANK repeats with a variety of ion channels/pumps, calcium release channels and cell adhesion molecules. Recent studies in mice indicate that ankyrins have a physiological role in restricting voltage-gated sodium channels and members of the L1 CAM family of cell adhesion molecules to excitable membranes in the central nervous system and in targeting calcium-release channels to the calcium homeostasis compartment of striated muscle.

LAD-1, the Caenorhabditis elegans L1CAM homologue, participates in embryonic and gonadal morphogenesis and is a substrate for fibroblast growth factor receptor pathway-dependent phosphotyrosine-based signaling

This study shows that L1-like adhesion (LAD-1), the sole Caenorhabditis elegans homologue of the L1 family of neuronal adhesion molecules, is required for proper development of the germline and the early embryo and embryonic and gonadal morphogenesis. In addition, the ubiquitously expressed LAD-1, which binds to ankyrin-G, colocalizes with the C. elegans ankyrin, UNC-44, in multiple tissues at sites of cell–cell contact. Finally, we show that LAD-1 is phosphorylated in a fibroblast growth factor receptor (FGFR) pathway-dependent manner on a tyrosine residue in the highly conserved ankyrin-binding motif, FIGQY, which was shown previously to abolish the L1 family of cell adhesion molecule (L1CAM) binding to ankyrin in cultured cells. Immunofluorescence studies revealed that FIGQY-tyrosine–phosphorylated LAD-1 does not colocalize with nonphosphorylated LAD-1 or UNC-44 ankyrin but instead is localized to sites that undergo mechanical stress in polarized epithelia and axon–body wall muscle junctions. These findings suggest a novel ankyrin-independent role for LAD-1 related to FGFR signaling. Taken together, these results indicate that L1CAMs constitute a family of ubiquitous adhesion molecules, which participate in tissue morphogenesis and maintaining tissue integrity in metazoans.

The C. elegans L1CAM homologue LAD-2 functions as a coreceptor in MAB-20/Sema2–mediated axon guidance

The L1 cell adhesion molecule (L1CAM) participates in neuronal development. Mutations in the human L1 gene can cause the neurological disorder CRASH (corpus callosum hypoplasia, retardation, adducted thumbs, spastic paraplegia, and hydrocephalus). This study presents genetic data that shows that L1-like adhesion gene 2 (LAD-2), a Caenorhabditis elegans L1CAM, functions in axon pathfinding. In the SDQL neuron, LAD-2 mediates dorsal axon guidance via the secreted MAB-20/Sema2 and PLX-2 plexin receptor, the functions of which have largely been characterized in epidermal morphogenesis. We use targeted misexpression experiments to provide in vivo evidence that MAB-20/Sema2 acts as a repellent to SDQL. Coimmunoprecipitation assays reveal that MAB-20 weakly interacts with PLX-2; this interaction is increased in the presence of LAD-2, which can interact independently with MAB-20 and PLX-2. These results suggest that LAD-2 functions as a MAB-20 coreceptor to secure MAB-20 coupling to PLX-2. In vertebrates, L1 binds neuropilin1, the obligate receptor to the secreted Sema3A. However, invertebrates lack neuropilins. LAD-2 may thus function in the semaphorin complex by combining the roles of neuropilins and L1CAMs.

Evaluation of highly accelerated simultaneous multi-slice EPI for fMRI

Echo planar imaging (EPI) is the MRI technique that is most widely used for blood oxygen level-dependent (BOLD) functional MRI (fMRI). Recent advances in EPI speed have been made possible with simultaneous multi-slice (SMS) methods which combine acceleration factors M from multiband (MB) radiofrequency pulses and S from simultaneous image refocusing (SIR) to acquire a total of N=S×M images in one echo train, providing up to N times speed-up in total acquisition time over conventional EPI. We evaluated accelerations as high as N=48 using different combinations of S and M which allow for whole brain imaging in as little as 100ms at 3T with a 32 channel head coil. The various combinations of acceleration parameters were evaluated by tSNR as well as BOLD contrast-to-noise ratio (CNR) and information content from checkerboard and movie clips in fMRI experiments. We found that at low acceleration factors (N≤6), setting S=1 and varying M alone yielded the best results in all evaluation metrics, while at acceleration N=8 the results were mixed using both S=1 and S=2 sequences. At higher acceleration factors (N>8), using S=2 yielded maximal BOLD CNR and information content as measured by classification of movie clip frames. Importantly, we found significantly greater BOLD information content using relatively fast TRs in the range of 300ms-600ms compared to a TR of 2s, suggesting that faster TRs capture more information per unit time in task based fMRI.

TRPM Channels Modulate Epileptic-like Convulsions via Systemic Ion Homeostasis

Neuronal networks operate over a wide range of activity levels, with both neuronal and nonneuronal cells contributing to the balance of excitation and inhibition. Activity imbalance within neuronal networks underlies many neurological diseases, such as epilepsy. The Caenorhabditis elegans locomotor circuit operates via coordinated activity of cholinergic excitatory and GABAergic inhibitory transmission. We have previously shown that a gain-of-function mutation in a neuronal acetylcholine receptor, acr-2(gf), causes an epileptic-like convulsion behavior. Here we report that the behavioral and physiological effects of acr-2(gf) require the activity of the TRPM channel GTL-2 in nonneuronal tissues. Loss of gtl-2 function does not affect baseline synaptic transmission but can compensate for the excitation-inhibition imbalance caused by acr-2(gf). The compensatory effects of removing gtl-2 are counterbalanced by another TRPM channel, GTL-1, and can be recapitulated by acute treatment with divalent cation chelators, including those specific for Zn(2+). Together, these data reveal an important role for ion homeostasis in the balance of neuronal network activity and a novel function of nonneuronal TRPM channels in the fine-tuning of this network activity.

CRASHing with the worm: Insights into L1CAM functions and mechanisms

The L1 family of cell adhesion molecules (L1CAMs) in vertebrates has long been studied for its roles in nervous system development and function. Members of this family have been associated with distinct neurological disorders that include CRASH, autism, 3p syndrome, and schizophrenia. The conservation of L1CAMs in Drosophila and Caenorhabditis elegans allows the opportunity to take advantage of these simple model organisms and their accessible genetic manipulations to dissect L1CAM functions and mechanisms of action. This review summarizes the discoveries of L1CAMs made in C. elegans, showcasing this simple model organism as a powerful system to uncover L1CAM mechanisms and roles in healthy and diseased states. Developmental Dynamics 239:1490–1501, 2010.

unc-44 Ankyrin and stn-2 γ-Syntrophin Regulate sax-7 L1CAM Function in Maintaining Neuronal Positioning in Caenorhabditis elegans

The L1 family of single-pass transmembrane cell adhesion molecules (L1CAMs) is conserved from Caenorhabditis elegans and Drosophila to vertebrates and is required for axon guidance, neurite outgrowth, and maintenance of neuronal positions. The extracellular region of L1CAMs mediates cell adhesion via interactions with diverse cell-surface and extracellular matrix proteins. In contrast, less is known regarding the function of the intracellular domains in the L1CAM cytoplasmic tail. Previously, we identified a role of the C. elegans L1CAM homolog, SAX-7, in maintaining neuronal and axonal positioning. Here, we demonstrate that this function is dependent on three conserved motifs that reside in the SAX-7 cytoplasmic tail: (1) the FERM-binding motif, (2) the ankyrin-binding domain, and (3) the PDZ-binding motif. Furthermore, we provide molecular and genetic evidence that UNC-44 ankyrin and STN-2 γ-syntrophin bind SAX-7 via the respective ankyrin-binding and PDZ-binding motifs to regulate SAX-7 function in maintaining neuronal positioning.

Caenorhabditis elegans reveals a FxNPxY-independent low-density lipoprotein receptor internalization mechanism mediated by epsin1

A genome-wide RNA interference screen using Caenorhabditis elegans LRP-1/megalin as a model for LDLR transport was employed to identify factors critical to LDLR uptake. We provide evidence that epsin1 promotes LDLR internalization via a FxNPxY-independent pathway. We complement C. elegans in vivo approaches with loss-of-function and biochemical analyses, using mammalian cell culture systems to evaluate epsin1’s mode of action in LDLR endocytosis.

C. elegans NIMA-related kinases NEKL-2 and NEKL-3 are required for the completion of molting.

Caenorhabditis elegans molting is a process during which the apical extracellular matrix of the epidermis, the cuticle, is remodeled through a process of degradation and re-synthesis. Using a genetic approach, we identified nekl-3 as essential for the completion of molting. NEKL-3 is highly similar to the mammalian NEK kinase family members NEK6 and NEK7. Animals homozygous for a hypomorphic mutation in nekl-3, sv3, had a novel molting defect in which the central body region, but not the head or tail, was unable to shed the old cuticle. In contrast, a null mutation in nekl-3, gk506, led to complete enclosure within the old cuticle. nekl-2, which is most similar to mammalian NEK8, was also essential for molting. Mosaic analyses demonstrated that NEKL-2 and NEKL-3 were specifically required within the large epidermal syncytium, hyp7, to facilitate molting. Consistent with this, NEKL-2 and NEKL-3 were expressed at the apical surface of hyp7 where they localized to small spheres or tubular structures. Inhibition of nekl-2, but not nekl-3, led to the mislocalization of LRP-1/megalin, a cell surface receptor for low-density lipoprotein (LDL)-binding proteins. In addition, nekl-2 inhibition led to the mislocalization of several other endosome-associated proteins. Notably, LRP-1 acts within hyp7 to facilitate completion of molting, suggesting at least one mechanism by which NEKL-2 may influence molting. Notably, our studies failed to reveal a requirement for NEKL-2 or NEKL-3 in cell division, a function reported for several mammalian NEKs including NEK6 and NEK7. Our findings provide the first genetic and in vivo evidence for a role of NEK family members in endocytosis, which may be evolutionarily conserved.

Neural integrity is maintained by dystrophin in C. elegans

The dystrophin protein complex, an important regulator of muscle membrane integrity, also maintains neural organization through interactions with the L1CAM family member SAX-7.