Dissection of the molecular machinery of micro- and macronucleophagy

Abstract

A highly conserved pathway required for cellular homeostasis, autophagy facilitates degradation and recycling of various cellular components. Involvement of autophagy in medical context has made associated research a highly relevant topic for a variety of diseases and a topic of interest for the development of novel therapeutical approaches. Nucleophagy, a functional subtype to autophagy, tasked with removal of portions of the nucleus has recently emerged as a pathway associated with various disease conditions, including neurodegenerative diseases, multiple cancer types and autoimmune diseases. Two principal modes of nucleophagy have been described: Macronucleophagy, a selective form of macroautophagy and micronucleophagy, represented by piecemeal microautophagy of the nucleus (PMN), which involves envelopment of nuclear material, nuclear envelope and portions of the nucleolus in vacuolar arms, pinching off a portion of nucleus into the vacuolar lumen. Nuclear degradation by PMN involves a membrane contact site of nucleus and vacuole, the nucleus vacuole junction (NVJ). With the increasing evidence that supports a role for compartmental contact sites as central hubs of cellular organisation, PMN could serve as a regulatory mechanism for the NVJ that displays a unique composition and is implicated in various processes of lipid metabolism and membrane regulation. Identification of Atg39 as the cargo receptor for perinuclear ER and the subsequent formulation of macronucleophagy has revised the perception of nucleophagy, previously thought to be represented by PMN. Parallels in cargo composition and a shared requirement for the core autophagic machinery !1 prompted investigations towards differentiating and further characterising the systems that facilitate nucleophagy. Data presented in this work suggests NVJ resident protein Nvj1 as the most reliable cargo for measuring exclusive degradation by PMN. Atg39, previously thought only to be required as the cargo receptor for macronucleophagy, was shown to be involved in PMN as well. The mechanism of PMN was further expanded by the discovery of a newly identified microautophagic membrane, present between the vacuolar arms that abstract the nuclear portion. Bulging of the nucleus, the initial step of PMN, was shown to be associated with an actin containing structure, visually reminiscent of a contractile ring. Data presented in this work supports a role for PMN as a regulatory function for the NVJ and downstream lipid metabolic processes rather than a clearance mechanism for nuclear material. Investigations of the macronucleophagic process produced novel insights into selective macroautophagy, detailing the three dimensional morphology of autophagosome formation under native conditions and discovery of alterations in membrane composition at the vacuolar autophagosomal contact site.