Richard L. Roberts

Evidence for a symmetrical requirement for Rab5-GTP in in vitro endosome-endosome fusion

Early endosome fusion, which has been extensively characterized using an in vitro reconstitution assay, is Rab5-dependent. To examine the requirement for Rab5 on both fusion partners, we prepared cytosol and endosomes depleted of Rab5. Unlike control cytosol, Rab5-depleted cytosol was only marginally active in the in vitro endosome fusion. However, fusion could be restored by the addition of wild-type Rab5 or Rab5 D136N, a mutant whose nucleotide specificity favors xanthine over guanine. The addition of Rab5 D136N restored fusion only in the presence of XTP. In the absence of XTP or in the presence of XDP, Rab5 D136N failed to restore fusion. When fusion was carried out with endosomal vesicles depleted of Rab GTPases (by preincubation of vesicles with GDP dissociation inhibitor), together with cytosol immunodepleted of Rab5, fusion was virtually absent. We then used immunodepleted cytosol and GDP dissociation inhibitor-treated vesicles to determine whether Rab5 is required by both fusion partners. Using separate sets of endosomal vesicles, we found that priming both sets of Rab5-depleted vesicles with Rab5 Q79L, a GTPase-defective mutant, substantially stimulated endosome fusion. Priming one set of vesicles with Rab5 Q79L and a second set of vesicles with Rab5 S34N failed to activate fusion. When both sets of Rab5-depleted vesicles were primed with Rab5 D136N supplemented with XTP, endosome fusion was stimulated, similar to that observed with Rab5 Q79L. However, when one set of vesicles was preincubated with Rab5 D136N plus XTP and the second set with Rab5 D136N and XDP, no stimulation of fusion was observed. We conclude that Rab5-GTP is required on both fusion partners for docking and fusion of early endosomes. To confirm the fusion of Rab5-GTP-positive vesiclesin vivo, we expressed GFP-Rab5 Q79L in fibroblasts and observed fusion of Rab5-positive vesicles. We failed to record fusion of Rab5-positive vesicles with Rab5-negative vesicles. We conclude that Rab5-GTP is required on both sets of endosomes for fusion in vitro and in living cells.

Familial colloid cyst of the third ventricle: case report and review of associated conditions.

The cases of a father and his son who were diagnosed with pathologically confirmed colloid cysts of the third ventricle are presented. The familial occurrence of this tumor is rare and suggests that genetic factors may play a role in its formation. Consistent with the concept that the cyst originates as a developmental abnormality, it is associated with a variety of congenital defects. The conditions that are associated with these tumors are discussed.

Structural and Functional Optical Imaging of Angiogenesis in Animal Models

Publisher Summary This chapter describes the structural and functional optical imaging of angiogenesis in animal models. Two animal models for microscopic optical imaging of angiogenesis are presented. Imaging studies have identified multiple cellular and molecular abnormalities that distinguish tumor vessels from their normal vessels and help explain their unusual appearance, irregular blood flow, and vascular leakiness. Most tumor vessels display a disorganized pattern and do not fit into the conventional hierarchy of arterioles, capillaries, and venules. The microphotographs of live tumors in the window chamber are used to obtain measurements of tumor vascular length density as an indicator of tumor vasculature. Vascularized tumors developed in 1 week, at the time they were treated with local irradiation. Irradiation treatment induced a dose- and time-dependent destruction to tumor blood vessels within the window. Radiation-sensitive tumor vessels in melanoma B16F0 showed a rapid and marked regression following 3 Gy of irradiation. It is suggested that optical imaging not only provides powerful tools with which to study the molecular mechanisms of tumor angiogenesis, identify therapeutic targets, and validate the targets in animal models, but it will also be a valuable measurement for monitoring drug response in patients.

16 - Measurement of Rab5 Protein Kinase B/akt and Regulation of Ras-Activated Endocytosis

This chapter describes the methodology to express the small GTPases, Rab5 and H-Ras, and the regulatory kinase, PKB/akt, using the Sindbis virus transient expression system for biochemical and morphological analysis of the endocytic pathway. Functional data are also provided to assess the physiologic relevance of the GTP-binding proteins. Because preparation of recombinant virus stocks and the expression Sindbis virus encoding cDNAs share identical procedures, the method is described only for the Rab5 recombinant Sindbis virus. In this method, cDNAs of Rab5 are subcloned into the unique XbaI site of the Sindbis vector. The plasmid is then linearized by cutting with XhoI. This cut DNA is used as template for the in vitro transcription. The resulting RNA transcripts are used for transfection of confluent BHK-21 cell monolayers using a lipofectin-mediated procedure. It is suggested that cells expressing Rab GTPases (H-Ras or PKB/akt) can be analyzed in a number of ways, including immunofluorescence confocal microscopy, electron microscopy, Western blot analysis, metabolic labeling, and measurements of virus processing during intracellular transport.