Theodore G. Drivas

A Network of Nuclear Envelope Membrane Proteins Linking Centromeres to Microtubules

In the fission yeast S. pombe, nuclei are actively positioned at the cell center by microtubules. Here, we show that cytoplasmic microtubules are mechanically coupled to the nuclear heterochromatin through proteins embedded in the nuclear envelope. This includes an integral outer nuclear membrane protein of the KASH family (Kms2) and two integral inner nuclear membrane proteins, the SUN-domain protein Sad1 and the previously uncharacterized protein Ima1. Ima1 specifically binds to heterochromatic regions and promotes the tethering of centromeric DNA to the SUN-KASH complex. In the absence of Ima1, or in cells harboring mutations in the centromeric Ndc80 complex, inefficient coupling of centromeric heterochromatin to Sad1 leads to striking defects in the ability of the nucleus to tolerate microtubule-dependent forces, leading to changes in nuclear shape, loss of spindle pole body components from the nuclear envelope, and partial dissociation of SUN-KASH complexes. This work highlights a framework for communication between cytoplasmic microtubules and chromatin.

Existence of a novel clathrin-independent endocytic pathway in yeast that depends on Rho1 and formin

Much like mammalian cells, yeast contain a Rho-dependent pathway for endocytosis in addition to canonical clathrin-dependent endocytosis.

Interaction between Epsin/Yap180 Adaptors and the Scaffolds Ede1/Pan1 Is Required for Endocytosis

The spatial and temporal regulation of the interactions among the approximately 60 proteins required for endocytosis is under active investigation in many laboratories. We have identified the interaction between monomeric clathrin adaptors and endocytic scaffold proteins as a critical prerequisite for the recruitment and/or spatiotemporal dynamics of endocytic proteins at early and late stages of internalization. Quadruple deletion yeast cells (DeltaDeltaDeltaDelta) lacking four putative adaptors, Ent1/2 and Yap1801/2 (homologues of epsin and AP180/CALM proteins), with a plasmid encoding Ent1 or Yap1802 mutants, have defects in endocytosis and growth at 37 degrees C. Live-cell imaging revealed that the dynamics of the early- and late-acting scaffold proteins Ede1 and Pan1, respectively, depend upon adaptor interactions mediated by adaptor asparagine-proline-phenylalanine motifs binding to scaffold Eps15 homology domains. These results suggest that adaptor/scaffold interactions regulate transitions from early to late events and that clathrin adaptor/scaffold protein interaction is essential for clathrin-mediated endocytosis.

Disruption of CEP290 microtubule/membrane-binding domains causes retinal degeneration

Mutations in the gene centrosomal protein 290 kDa (CEP290) cause an array of debilitating and phenotypically distinct human diseases, ranging from the devastating blinding disease Leber congenital amaurosis (LCA) to Senior-Løken syndrome, Joubert syndrome, and the lethal Meckel-Gruber syndrome. Despite its critical role in biology and disease, very little is known about CEP290s function. Here, we have identified 4 functional domains of the protein. We found that CEP290 directly binds to cellular membranes through an N-terminal domain that includes a highly conserved amphipathic helix motif and to microtubules through a domain located within its myosin-tail homology domain. Furthermore, CEP290 activity was regulated by 2 autoinhibitory domains within its N and C termini, both of which were found to play critical roles in regulating ciliogenesis. Disruption of the microtubule-binding domain in a mouse model of LCA was sufficient to induce significant deficits in cilium formation, which led to retinal degeneration. These data implicate CEP290 as an integral structural and regulatory component of the cilium and provide insight into the pathological mechanisms of LCA and related ciliopathies. Further, these data illustrate that disruption of particular CEP290 functional domains may lead to particular disease phenotypes and suggest innovative strategies for therapeutic intervention.

Basal exon skipping and genetic pleiotropy: A predictive model of disease pathogenesis.

Basal exon skipping, a mechanism by which a cell compensates for deleterious mutations, explains pleiotropic disease pathogenesis in patients with mutations in two different ciliopathy-associated genes. A new model of disease pleiotropy To investigate a potential mechanism explaining genetic pleiotropy, Drivas et al. correlated the genotype/phenotype information of individuals affected with two different autosomal recessive ciliopathies to the molecular biologic parameters in their cells. In both cases, disease severity correlated with amounts of the relevant protein. Basal exon skipping explains the diverse phenotypes seen in these different ciliopathies and may also explain the pathologic findings associated with other inherited disorders. Genetic pleiotropy, the phenomenon by which mutations in the same gene result in markedly different disease phenotypes, has proven difficult to explain with traditional models of disease pathogenesis. We have developed a model of pleiotropic disease that explains, through the process of basal exon skipping, how different mutations in the same gene can differentially affect protein production, with the total amount of protein produced correlating with disease severity. Mutations in the centrosomal protein of 290 kDa (CEP290) gene are associated with a spectrum of phenotypically distinct human diseases (the ciliopathies). Molecular biologic examination of CEP290 transcript and protein expression in cells from patients carrying CEP290 mutations, measured by quantitative polymerase chain reaction and Western blotting, correlated with disease severity and corroborated our model. We show that basal exon skipping may be the mechanism underlying the disease pleiotropy caused by CEP290 mutations. Applying our model to a different disease gene, CC2D2A (coiled-coil and C2 domains–containing protein 2A), we found that the same correlations held true. Our model explains the phenotypic diversity of two different inherited ciliopathies and may establish a new model for the pathogenesis of other pleiotropic human diseases.

CEP290 and the primary cilium.

The protein CEP290 has recently emerged as a major player in the biology of the cilium and as a causative protein in a number of human syndromic diseases, most of which are associated with the devastating blinding disease Leber congenital amaurosis. (Coppieters et al., Hum Mutat 31, 2010, 1097-1108) CEP290 is known to be an important component of the primary cilium, localizing to the Y-links of the ciliary transition zone and having a role in the regulation of transport in and out of the ciliary compartment (Craige et al., J Cell Biol 190, 2010, 927-940). While many mutations in CEP290 have been identified in human patients, how these mutations result in the spectrum of human disease attributed to the protein remain unknown. As we begin to learn more about the normal role of CEP290, it is likely that we will begin to shed light on how these mutations result in the various CEP290 disease phenotypes. Here we discuss many of these diverse aspects of CEP290 biology and pathology in an attempt to link what we know about the molecular mechanisms of CEP290 function with what we know about CEP290-associated disease.

An Autogenously Regulated Expression System for Gene Therapeutic Ocular Applications

The future of treating inherited and acquired genetic diseases will be defined by our ability to introduce transgenes into cells and restore normal physiology. Here we describe an autogenous transgene regulatory system (ARES), based on the bacterial lac repressor, and demonstrate its utility for controlling the expression of a transgene in bacteria, eukaryotic cells, and in the retina of mice. This ARES system is inducible by the small non-pharmacologic molecule, Isopropyl β-D-1-thiogalactopyranoside (IPTG) that has no off-target effects in mammals. Following subretinal injection of an adeno-associated virus (AAV) vector encoding ARES, luciferase expression can be reversibly controlled in the murine retina by oral delivery of IPTG over three induction-repression cycles. The ability to induce transgene expression repeatedly via administration of an oral inducer in vivo, suggests that this type of regulatory system holds great promise for applications in human gene therapy.

Existence of a Novel Clathrin-Independent Endocytic Pathway in Yeast

Yeast is a powerful model organism for dissecting the temporal stages and choreography of the complex protein machinery during endocytosis. The only known mechanism for endocytosis in yeast is clathrin-mediated endocytosis, even though clathrin-independent endocytic pathways have been described in other eukaryotes. In my talk, I will present evidence for a clathrin-independent endocytic pathway in yeast. In cells lacking the clathrin-binding adaptor proteins Ent1, Ent2, Yap1801 and Yap1802, we identified a second endocytic pathway that depends on the GTPase Rho1, the downstream formin Bni1, and the Bni1 co-factors Bud6 and Spa2. This second pathway does not require components of the better-studied endocytic pathway, including clathrin and Arp2/3 complex activators. Thus, our results reveal the existence of a second pathway for endocytosis in yeast, suggesting similarities with the RhoA-dependent endocytic pathways of mammalian cells.