CHARACTERIZATION OF A NOVEL MICROTUBULE BINDING PROTEIN CLIPR76 Abstract by Gergana T. Ugrinova Eukaryotic cells are highly compartmentalized and spatially organized. In order to maintain the appropriate distribution of organelles, cells use various mechanisms for transport. Trafficking of vesicles and/or organelles to specific locations throughout the cell is a complex process involving a number of molecules. This transport is primarily driven by molecular motors tracking along microtubules and microfilaments. Motor proteins recognize the inherent microtubule polarity and move preferentially to either minus or plus end. Although significant progress has been made in characterizing molecular motor machinery, exactly how cells control motor activities to achieve synchronized movement and proper targeting of the cargoes is yet unclear. In recent years, extensive research to identify molecules that interact with membrane organelles and microtubules has led to the discovery of an array of non-motor microtubule associated proteins, which serve to connect motors with their cognate cargoes. These proteins are called cytoplasmic linker proteins (CLIPs). A family of CLIPs with founding member CLIP-170 shares the ability to bind microtubules via conserved CAPGly domains. Some members of the CLIP-170 related (Ì¢åÛåÏClipRÌ¢åÛå ) family interact specifically with distinct membranous organelles. Thus, characterization of new ClipR Gergana T. Ugrinova proteins would expand our knowledge of the proteins that mediate and regulate membrane-microtubule interactions. A combination of cell biological, molecular biological and biochemical analyses was used to study a novel member of ClipR family, CLIPR76. Sequence analysis and experimental evidence show that the CLIPR76 gene is alternatively spliced and produces multiple isoforms in different organisms. Western blot and real time PCR investigations show that the CLIPR76 gene is ubiquitously expressed with highest levels in testis and striated muscle. Some isoforms appear to be tissue specific. Immunofluorescence microscopy of the GFP tagged or untagged CLIPR76 proteins demonstrate that the different isoforms have different cellular localization and imply that they have different protein activities. Some CLIPR76 isoforms localize to the endoplasmic reticulum and their overexpression affect the morphology of ER and ERGIC membrane compartments, while others localize to microtubules or subsets of microtubules and cause alterations in microtubule organization when expressed at higher levels. These results imply a role for CLIPR76 proteins in membrane transport and ER microtubules interactions.