Microtubule-dependent formation of the stigmoid body as a cytoplasmic inclusion distinct from pathological aggresomes

Abstract

The stigmoid body (STB) is a neurocytoplasmic inclusion containing huntingtin-associated protein 1 (HAP1), an interactor of huntingtin, and its formation is induced by transfection of HAP1-cDNA into cultured cells. Although STB is believed to play a protective role in polyglutamine diseases, including Huntington's disease and spinal and bulbar muscular atrophy, by sequestering the causative proteins, huntingtin and androgen receptor, respectively, its physiological function and formation remain poorly understood. Therefore, STB is occasionally confused with another cytoplasmic inclusion observed in polyglutamine diseases, the aggresome. Here we examined the subcellular dynamics of STB and compared it immunohistochemically and cytochemically with the aggresome in the rat brain and COS-7 or HeLa cells transfected with HAP1 and/or polyglutamine disease-associated genes. In time-lapse image analysis of HAP1-transfected cells, the HAP1-induced STB is formed from multiple fusions of small HAP1 inclusions characterized by vigorous cytoplasmic movement. In HAP1-transfected cells treated with a microtubule-depolymerizing drug, although the formation of small HAP1 inclusions was not affected, their fusion was critically inhibited. Immunohistochemistry and cytochemistry revealed the absence of association between STB and aggresomal markers, such as ubiquitin/proteasome, intermediate filaments, and the centrosome. Taken together, we concluded that STB is formed by a two-step process comprising microtubule-independent formation of small HAP1 inclusions and microtubule-dependent fusion of these inclusions, and that STB is distinct from pathological aggresomes.