Characterization of the In Vivo Targeting of Cytoplasmic Dynein

Cytoplasmic dynein is a minus-end-directed microtubule motor protein that uses ATP and an accessory complex, dynactin, to move vesicles along intracellular microtubule frameworks. In neurons, it is required for moving vesicles at neuronal synaptic terminals back to the cell body, a process termed "retrograde transport". Cytoplasmic dynein has also been implicated in other cellular activities, such as spindle assembly, formation and positioning of the Golgi complex, proper distribution of the endosomes and lysosomes in mammalian cells and nuclear migration in fungi. Although cytoplasmic dynein is involved in multiple cellular functions, little is known about how its intracellular targeting is regulated. The filamentous fungus Aspergillus nidulans is an excellent genetic model system to study the in vivo function and regulation of cytoplasmic dynein. In A. nidulans, cytoplasmic dynein is required for the process of nuclear migration along the growing hyphae. Components involved in cytoplasmic dynein function have been and will continuously be isolated as the nud (nuclear distribution) gene products. Several of the nud genes we have previously characterized encode subunits of the cytoplasmic dynein and dynactin complexes. For example, the nudA gene encodes the heavy chain of cytoplasmic dynein that contains the motor activity. Potential regulators of cytoplasmic dynein not in the dynein/dynactin complexes have also been isolated, which include the nudF gene product that is homologous to the human protein LIS1 required for neuronal migration during brain development. Recently, the investigator has made an exciting observation that both NUDA (cytoplasmic dynein heavy chain) and NUDF (LIS-1-like protein) localize to the dynamic ends of microtubules. This observation was made in living cells by using the green fluorescent protein (GFP) tagged NUDA and NUDF constructs that are functional in vivo. How NUDA and NUDF get targeted to this cellular location is an important cell biological question. To identify which nud gene products are required for the localization of NUDA and NUDF to microtubule ends, the nudK gene that encodes the Arp1 (the actin related protein) protein in the dynactin complex, and the nudI gene, which encodes an intermediate chain of cytoplasmic dynein were cloned. The in vivo localization as well as the requirement for other NUD proteins will be investigated. Finally, other gene products which may regulate the function of cytoplasmic dynein will be sought out. Results obtained from these studies will be analyzed in relationship with the microtubule-end-targeting of NUDA or NUDF to reveal novel aspects of dynein/dynactin and NUDF targeting in vivo. This work to be carried in a fungal model system will potentially lead to insights of general significance into the control of dynein.