Timothy Evans

CDX2 , a human homologue of Drosophila caudal , is mutated in both alleles in a replication error positive colorectal cancer

The Cdx2 gene is one of three murine homologues of the Drosophila homeobox gene caudal. Mice heterozygous for a null mutation in Cdx2 exhibit a variable phenotype including tail abnormalities, stunted growth and a homeotic shift of vertebrae. Most strikingly, however, 90% of heterozygous mice were reported to develop multiple intestinal adenomatous polyps, most notably in the proximal colon (). These observations led us to propose that mutation of CDX2 may be involved in the genesis of some human colorectal tumours. A survey of DNA from 85 colorectal tumours revealed that one with extensive microsatellite instability (RER+ phenotype) has mutations in both alleles of CDX2. Both mutations occur in coding regions which contain repetitive elements and are consistent with those found in RER+ tumours.

Rab23, a negative regulator of hedgehog signaling, localizes to the plasma membrane and the endocytic pathway.

The regulation of hedgehog signaling by vesicular trafficking was exemplified by the finding that Rab23, a Rab‐GTPase vesicular transport protein, is mutated in open brain mice. In this study, the localization of Rab23 was analyzed by light and immunoelectron microscopy after expression of wild‐type (Rab23‐GFP), constitutively active Rab23 (Rab23Q68L‐GFP), and inactive Rab23 (Rab23S23N‐GFP) in a range of mammalian cell types. Rab23‐GFP and Rab23Q68L‐GFP were predominantly localized to the plasma membrane but were also associated with intracellular vesicular structures, whereas Rab23S23N‐GFP was predominantly cytosolic. Vesicular Rab23‐GFP colocalized with Rab5Q79L and internalized transferrin‐biotin, but not with a marker of the late endosome or the Golgi complex. To investigate Rab23 with respect to members of the hedgehog signaling pathway, Rab23‐GFP was coexpressed with either patched or smoothened. Patched colocalized with intracellular Rab23‐GFP but smoothened did not. Analysis of patched distribution by light and immunoelectron microscopy revealed it is primarily localized to endosomal elements, including transferrin receptor‐positive early endosomes and putative endosome carrier vesicles and, to a lesser extent, with LBPA‐positive late endosomes, but was excluded from the plasma membrane. Neither patched or smoothened distribution was altered in the presence of wild‐type nor mutant Rab23‐GFP, suggesting that despite the endosomal colocalization of Rab23 and patched, it is likely that Rab23 acts more distally in regulating hedgehog signaling.

A Novel Hook-Related Protein Family and the Characterization of Hook-Related Protein 1

The spatial organization of organelles within a cell is dependent on microtubules. Recently, members of the Hook family of proteins have been proposed to function in linking organelles to microtubules. We report the identification of a completely novel protein family, the Hook‐related protein (HkRP) family, from which the Hook proteins have diverged. Bioinformatic analysis of the HkRP family revealed several conserved domains, including a unique C‐terminal HkRP domain. The central region of each protein is comprised of an extensive coiled‐coil domain, and the N‐terminus contains a putative microtubule‐binding domain. This domain has been shown to bind microtubules in the Hook protein and show that the HkRP1 protein is microtubule‐associated. While endogenous HkRP1 has no distinct organelle association, expression of the C‐terminal membrane‐binding domain suggests a function of the HkRP1 in early endosome. Ultrastructural studies reveal that expression of the C‐terminal HkRP1 domain causes an accumulation of internal membranes with an electron‐dense coat. Co‐localization studies show a concomitant redistribution of the early endosome marker sorting‐nexin 1 but not the early endosome antigen‐1 (EEA1). The steady‐state distribution of the epidermal growth factor receptor is also specifically disrupted by expression of the C‐terminal domain. We propose that HkRP1 is involved in the process of tubulation of sorting nexin‐1 positive membranes from early endosome subdomains.

No evidence for the H133Y mutation in SONIC HEDGEHOG in a collection of common tumour types

The human homologue of the Drosophila segment polarity gene patched is mutated in the cancer predisposition syndrome naevoid basal cell carcinoma syndrome (NBCCS), as well as in several types of tumour associated with the disorder. It was recently reported that a single recurrent mutation in the SONIC HEDGEHOG gene, which encodes the PATCHED ligand, was found in one of 43 basal cell carcinomas (BCCs), one of 14 medulloblastomas and one of six breast carcinomas analysed (. We have searched extensively for this same mutation in a large collection of BCCs, medulloblastomas and carcinomas of the breast, ovary and colorectum and have failed to detect the mutation in any sample analysed.

Clathrin promotes centrosome integrity in early mitosis through stabilization of centrosomal ch-TOG

Clathrin inactivation during S phase destabilizes the microtubule-binding protein ch-TOG, affecting its centrosomal localization and centrosome integrity during early mitosis.

Sequence variants of DLC1 in colorectal and ovarian tumours

Loss of heterozygosity occurs frequently on the short arm of chromosome 8 in many neoplasms, including colorectal and ovarian cancer. Monochromosome transfer experiments into colorectal tumour cell lines have provided functional evidence for a tumour suppressor gene located at 8p22–23. One of the genes from this region that is expressed by our suppressed hybrids is a candidate tumour suppressor gene, DLC1 (deleted in liver cancer), which has homology to rat RhoGAP. We have delineated the structure of the DLC1 gene and used single‐stranded conformation polymorphism analysis (SSCP) to look for sequence variants in 126 colorectal and 33 ovarian primary tumours and cell lines. One exonic missense mutation and three intronic insertions/deletions were identified in primary colorectal tumours, as well as many polymorphisms present in germline DNAs. The rarity of exonic missense mutations, and the absence of protein‐truncating mutations, indicates that DLC1 is not the target of 8p LOH in colorectal or ovarian tumours. The delineation of the gene structure allows mutation analysis of DLC1 in other tumour types for which it remains a candidate tumour suppressor gene based on its location and homology to rhoGAP. Hum Mutat 15:156–165, 2000.

The spectrum of patched mutations in a collection of Australian basal cell carcinomas

Inactivating mutations in the human patched (PTCH) gene have been identified in both familial and sporadic basal cell carcinomas (BCCs). In some tumors mutations have been detected in both alleles thereby supporting the role of PTCH as a tumor suppressor gene. We have analyzed 22/23 coding exons of PTCH for mutations in 44 sporadic BCCs, and detected 10 novel mutations in nine tumors. In two of the mutant tumors the remaining allele was inactivated by loss of heterozygosity. Five novel PTCH polymorphisms were also identified. Most of the variations found were C>T substitutions at dipyrimidine sites, supporting previous studies which indicate a role for ultraviolet‐B in the genesis of sporadic BCCs. Hum Mutat 16:43–48, 2000.

Characterization of Rab23, a negative regulator of sonic hedgehog signaling

The hedgehog signaling pathway is indispensable in embryogenesis, being responsible for the development of a wide array of vertebrate organs. Given its importance in embryogenesis, the precise regulation of hedgehog signaling is crucial. Aberrant activation of this pathway in postnatal life has been associated with a number of tumor types, reinforcing the role of developmental signaling pathways in tumorigenesis. The small GTPase Rab23 acts as a negative regulator of the hedgehog signaling pathway, most notably in the vertebrate neural system. By analogy with studies of other Rab proteins, analysis of the localization of wild-type and constitutively active and inactive forms of Rab23 provides the potential to shed light on the role of Rab23 at the cellular level. We previously produced expression constructs encoding these proteins for analysis in mammalian cell cultures at both the light and the electron microscopy level. This revealed that both wild-type and active Rab23 localizes to the plasma membrane and to endocytic vesicles (T. M. Evans et al. [2003] Traffic4, 869-884). We describe the methods used to design and make the Rab23 expression constructs, and to assess their localization relative to key hedgehog pathways and endocytic markers in both transiently and stably transfected cell cultures.

Clathrin light chains are required for the gyrating-clathrin recycling pathway and thereby promote cell migration

The clathrin light chain (CLC) subunits participate in several membrane traffic pathways involving both clathrin and actin, through binding the actin-organizing huntingtin-interacting proteins (Hip). However, CLCs are dispensable for clathrin-mediated endocytosis of many cargoes. Here we observe that CLC depletion affects cell migration through Hip binding and reduces surface expression of β1-integrin by interference with recycling following normal endocytosis of inactive β1-integrin. CLC depletion and expression of a modified CLC also inhibit the appearance of gyrating (G)-clathrin structures, known mediators of rapid recycling of transferrin receptor from endosomes. Expression of the modified CLC reduces β1-integrin and transferrin receptor recycling, as well as cell migration, implicating G-clathrin in these processes. Supporting a physiological role for CLC in migration, the CLCb isoform of CLC is upregulated in migratory human trophoblast cells during uterine invasion. Together, these studies establish CLCs as mediating clathrin–actin interactions needed for recycling by G-clathrin during migration.

Symptomatic Hypotonic Hyponatremia Presenting at High Altitude

We present a case of altered mental status and seizure that occurred at an altitude known to cause high altitude-related illnesses. Based on the presenting symptoms, the patient was initially transferred to the hospital with a presumptive diagnosis of high altitude cerebral edema. On review of imaging and laboratory data, she was found to be experiencing symptomatic hypotonic hyponatremia. This case presented an interesting diagnostic challenge and underscores the importance of maintaining a broad differential diagnosis when evaluating a patient with altered mental status from an alpine setting.