Yuh Nung Jan

Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence

A DNA binding and dimerization motif, with apparent amphipathic helices (the HLH motif), has recently been identified in various proteins, including two that bind to immunoglobulin enhancers (E12 and E47). We show here that various HLH proteins can bind as apparent heterodimers to a single DNA motif and also, albeit usually more weakly, as apparent homodimers. The HLH domain can mediate heterodimer formation between either daughterless, E12, or E47 (Class A) and achaete-scute T3 or MyoD (Class B) to form proteins with high affinity for the kappa E2 site in the immunoglobulin kappa chain enhancer. The achaete-scute T3 and MyoD proteins do not form kappa E2-binding heterodimers together, and no active complex with N-myc was evident. The formation of a heterodimer between the daughterless and achaete-scute T3 products may explain the similar phenotypes of mutants at these two loci and the genetic interactions between them. A role of E12 and E47 in mammalian development, analogous to that of daughterless in Drosophila, is likely.

A New ER Trafficking Signal Regulates the Subunit Stoichiometry of Plasma Membrane KATP Channels

Proper ion channel function often requires specific combinations of pore-forming alpha and regulatory beta subunits, but little is known about the mechanisms that regulate the surface expression of different channel combinations. Our studies of ATP-sensitive K+ channel (K(ATP)) trafficking reveal an essential quality control function for a trafficking motif present in each of the alpha (Kir6.1/2) and beta (SUR1) subunits of the K(ATP) complex. We show that this novel motif for endoplasmic reticulum (ER) retention/retrieval is required at multiple stages of K(ATP) assembly to restrict surface expression to fully assembled and correctly regulated octameric channels. We conclude that exposure of a three amino acid motif (RKR) can explain how assembly of an ion channel complex is coupled to intracellular trafficking.

A Trafficking Checkpoint Controls GABAB Receptor Heterodimerization

Surface expression of GABA(B) receptors requires heterodimerization of GB1 and GB2 subunits, but little is known about mechanisms that ensure efficient heterodimer assembly. We found that expression of the GB1 subunit on the cell surface is prevented through a C-terminal retention motif RXR(R); this sequence is reminiscent of the ER retention/retrieval motif RKR identified in subunits of the ATP-sensitive K+ channel. Interaction of GB1 and GB2 through their C-terminal coiled-coil alpha helices masks the retention signal in GB1, allowing the plasma membrane expression of the assembled complexes. Because individual GABA(B) receptor subunits and improperly assembled receptor complexes are not functional even if expressed on the cell surface, we conclude that a trafficking checkpoint ensures efficient assembly of functional GABA(B) receptors.

Control of Daughter Cell Fates during Asymmetric Division: Interaction of Numb and Notch

During development of the Drosophila peripheral nervous system, a sensory organ precursor (SOP) cell undergoes rounds of asymmetric divisions to generate four distinct cells of a sensory organ. Numb, a membrane-associated protein, is asymmetrically segregated into one daughter cell during SOP division and acts as an inherited determinant of cell fate. Here, we show that Notch, a transmembrane receptor mediated cell-cell communication, functions as a binary switch in cell fate specification during asymmetric divisions of the SOP and its daughter cells in embryogenesis. Moreover, numb negatively regulates Notch, probably through direct protein-protein interaction that requires the phosphotyrosine-binding (PTB) domain of Numb and either the RAM23 region or the very C-terminal end of Notch. Notch then positively regulates a transcription factor encoded by tramtrack (ttk). This leads to Ttk expression in the daughter cell that does not inherit Numb. Thus, the inherited determinant Numb bestows a bias in the machinery for cell-cell communication to allow the specification of distinct daughter cell fates.

Asymmetric cell division.

With the recent identification of intrinsic cell-fate determinants for asymmetric cell division in several systems, biologists have begun to gain insight into the cellular mechanisms by which these determinants are preferentially segregated into one of the two daughter cells during mitosis so that the daughter cells acquire different fates.

Hippocampal Neuronal Polarity Specified by Spatially Localized mPar3/mPar6 and PI 3-Kinase Activity

How a neuron becomes polarized remains an outstanding question. Here, we report that selection of the future axon among neurites of a cultured hippocampal neuron requires the activity of growth factor receptor tyrosine kinase, phosphatidylinositol 3-kinase (PI 3-kinase), as well as atypical protein kinase C (aPKC). The PI 3-kinase activity, highly localized to the tip of the newly specified axon of stage 3 neurons, is essential for the proper subcellular localization of mPar3, the mammalian homolog of C. elegans polarity protein Par3. Polarized distribution of not only mPar3 but also mPar6 is important for axon formation; ectopic expression of mPar6 or mPar3, or just the N terminus of mPar3, leaves neurons with no axon specified. Thus, neuronal polarity is likely to be controlled by the mPar3/mPar6/aPKC complex and the PI 3-kinase signaling pathway, both serving evolutionarily conserved roles in specifying cell polarity.

A protein component of Drosophila polar granules is encoded by vasa and has extensive sequence similarity to ATP-dependent helicases

Determinants of pole cells, which are precursors of the germ line, are provided maternally and are localized to the posterior pole of the Drosophila egg, as are polar granules. It has been hypothesized that certain RNA molecules associated with polar granules may be necessary for pole cell determination. Using a monoclonal antibody (Mab46F11) against polar granules, we have cloned the gene for one of their components. This gene turns out to be vasa, which is required maternally for the formation of polar granules and germ cells. This polar granule component shows significant sequence similarity to eIF-4A, a translation initiation factor that binds to mRNA, and to other helicases.

Properties of the larval neuromuscular junction in Drosophila melanogaster.

The anatomy and physiology of the Drosophila larval neuromuscular junction were studied. 2. The dependence of muscle resting potentials on [K+]o and [Na+]o follows the Goldman‐Hodgkin‐Katz equation (PNa/PK=0‐23). Chloride ions distribute passively across the membrane. 3. The mean specific membrane resistance of muscle fibres is 4‐3 X 10(3) omega cm2, and the mean specific membrane capacitance is 7‐1 muF/cm2. The muscle fibre is virtually isopotential. 4.Transmitter release is quantal. Both the miniature excitatory junctional potential and the evoked release follow the Poisson distribution. 5. Transmitter release depends on approximately the fourth power of [Ca2+]o. If Sr2+ replaces Ca2+, it depends on approximately the fourth power of [Sr2+]o. Mg2+ reduces transmitter release without altering the fourth power dependence on [Ca2+]o.

atonal is a proneural gene that directs chordotonal organ formation in the Drosophila peripheral nervous system

In the Drosophila peripheral nervous system, proneural genes of the achaete-scute complex (ASC) are required for formation of the precursors of external sense organs but not of chordotonal organs. We report the isolation of a gene, atonal (ato), with evidence that it is a proneural gene for the formation of chordotonal organs. This gene is expressed in the proneural clusters and sense organ precursors that give rise to the embryonic and adult chordotonal, but not external sense, organs. Chordotonal organs are eliminated in embryos carrying chromosomal deficiencies that remove ato. Like the ASC products, ato protein contains a basic-helix-loop-helix region and heterodimerizes with daughterless protein to bind to E boxes. Moreover, ectopic expression of ato promotes the formation of extra sense organs. Despite similar proneural properties, we find that ectopic expression of the ASC genes promotes external sense organ formation exclusively, whereas ato promotes chordotonal organ formation preferentially. Thus, proneural genes are major determinants of neuronal identity.

Asymmetric Localization of a Mammalian Numb Homolog during Mouse Cortical Neurogenesis

During Drosophila neurogenesis, differential segregation of Numb is necessary for daughter cells of asymmetric divisions to adopt distinct fates, at least partly by biasing the Notch-mediated cell-cell interaction. We have isolated a highly conserved mammalian homolog of Drosophila numb, m-numb. During mouse cortical neurogenesis, m-Numb is asymmetrically localized to the apical membrane of dividing ventricular neural progenitors. Depending upon the orientation of the cleavage plane, m-Numb may be distributed into one or both of the daughter cells. When expressed in Drosophila embryos, m-Numb is localized asymmetrically in dividing neural precursors and rescues the numb mutant phenotype. Furthermore, m-Numb can physically interact with mouse Notch1. We propose that some shared molecular mechanisms, both cell-intrinsic and cell-extrinsic, generate asymmetric cell divisions during neurogenesis of vertebrates and invertebrates.