The necessity of nucleophagic modality

ABSTRACT

Nucleophagy, the selective subtype of autophagy that predominantly targets only a selected and (nonessential) portion of the nucleus, and rarely the nucleus in its entirety, for degradation, reinforces the paradigm that nucleophagy recycling is a meticulous and highly delicate process guarded by fail-safe mechanisms. Our goal in this commentary is to encourage autophagy researchers and other scientists to explore nucleophagy blind spots and gain advanced insights into the diverse roles of this process and its selective modality as they pertain to intranuclear quality control and cellular homeostasis. Identifying and deciphering nucleophagic signaling, regulation, molecular mechanism(s) and its mediators, cargo composition and nuclear membrane dynamics under numerous physiological and/or pathological settings will provide important advances in our understanding of this critical type of organelle-selective autophagy.

Abbreviations: INM, inner nuclear membrane; LN, late nucleophagy; mRNA, messenger RNA; NE, nuclear envelope; NL, nuclear lamina; NPC(s), nuclear pore complex(es); NVJ(s), nucleus-vacuole junction(s); ONM, outer nuclear membrane; PMN, piecemeal microautophagy of the nucleus; PND, programmed nuclear death; PNuD, programmed nuclear destruction; rDNA/rRNA, ribosomal DNA/RNA

The highly organized nucleus, a most prominent, complex and dynamic double membrane-bound organelle containing most of the cellular genetic material is the quintessential morphological and functional feature of eukaryotes as it maintains the integrity of genes and controls diverse cellular activities (i.e., compartmentalization, metabolism, growth, survival, reproduction and proliferation) by regulating gene expression, spatially and temporally [1]. The function-specific and architecturally distinct components/compartments of the nucleus are: (1) the nucleic acids (regarded as the essential components of the nucleus); namely, the highly compact DNA molecules which are organized into chromatin and subsequently chromosomes, messenger RNA (mRNA), and ribosomal DNA (rDNA) and RNA (rRNA). (2) The nuclear envelope (NE), consisting of two bordering membranes (a ribosome-studded outer nuclear membrane [ONM] and inner nuclear membrane [INM]) separated by the perinuclear space, and joined at annular junctions by bilateral, large multinucleoporin gateways/channels known as nuclear pore complexes (NPCs). (3) The plurifunctional and multistructural nucleolus, nucleolar associated proteins and dot-like nucleolar bodies. (4) The nuclear matrix/nucleoskeleton (which is lacking in yeast cells) consisting of the network of fibers known as the nuclear lamina (NL; composed of intermediate filaments known as lamins and nuclear lamin-associated membrane proteins). (5) The nucleoplasm that either harbors most of the above-mentioned nuclear components (i.e., nucleic acids, nucleolus, nuclear matrix) or interacts with others (i.e., NE and NPCs) [1–7].

Given that the nucleus functions as the cell’s “brain” by safeguarding the genetic material in addition to controlling its expression via mRNA transcription and translation [1,2], it is crucial to maintain nuclear homeostasis and in particular degradation and recycling of the nuclear components in a timely, highly selective and tightly regulated manner, in the context of both the cell’s physiology and pathology. To maintain intranuclear quality control and nuclear homeostasis (turnover rate of diverse components of the nucleus), cells encountering diverse stress conditions require a delicate strategy for removing undesirable, damaged and aberrant nuclear components. The selective subtype of autophagy known as nucleophagy (an “umbrella term”) is an ideal and elegant degradation process that selectively degrades (and subsequently recycles) the nuclear components and even the nucleus in its entirety [3–10]. To date, numerous modes of nucleophagy have been observed in diverse study models and species, and current data indicate that nucleophagy can be principally divided into 4 primary modes: (1) macronucleophagy, (2) “classical micronucleophagy”, (3) nucleophagy of the entire nucleus (“complete nucleophagy”) and (4) unconventional nucleophagy. Moreover, due to their morphological, mechanistic and functional differences, each of those 4 primary modes can be further subdivided (Table 1) [3–10].

Table 1.

Four primary modes of nucleophagy

Nucleophagy mode	Nucleophagic cargo/substrates	Key characteristics
MODE 1: MACRONUCLEOPHAGY (mammalian cells): 1. Bulk macronucleophagy [11,13,15–17] 2. Micronuclear macronucleophagy [12] 3. Chromatophagy/chromatinophagy [14,18,19] MODE 1: MACRONUCLEOPHAGY (yeast Saccharomyces cerevisiae): 1. Atg39-dependent macronucleophagy [4,20,21] 2. NPC-phagy [22–24] 3. Nucleoporinophagy [22–24]	LMNA (lamin A/C) and EMD (emerin) Nuclear lamina components (e.g., hetrochromatin protein, lamin-associated chromatin domains, LMNA and LMNB1 [lamin B1]) Chromatin fragments budding off the nucleus NE-enclosed micronuclei containing damaged chromosomal fragments NE protrusions containing chromatin, LMNA ONM, INM, Hmg1/NE protein 1 and Src1/steroid receptor coactivator 1, nucleolar protein Nop1 NE fragments containing multiple NPCs and portions of the nucleoplasm Nup159 and/or Nup159-containing (nucleoporin) subcomplex, portion of the nucleoplasm	Pathological/stress-induced and/or disease-associated conditions (e.g., genotoxicity, cellular senescence, oncogenic insult, cell cycle perturbation). Perinuclear autophagosome and autolysosome formation. Autophagic machinery interaction. Blebbing of the nucleus and nuclear membrane rearrangements. Pathological/stress-induced and/or disease-associated conditions. Autophagosome and autolysosome formation. Atg5- Atg7-, Atg8-dependent. Blebbing of the nucleus and nuclear membrane rearrangements. Pathological/stress-induced conditions (i.e., arginine starvation of cancer cells). Giant autophagosome and autolysosome formation. DNA/chromatin leakage. Autophagic machinery interaction. Nuclear membrane rupture and/or rearrangements. Nutrient deprivation-induced conditions. Micronuclei formation. Autophagosome formation. Atg39-receptor mediated. Partial involvement of Atg40. Autophagic machinery requirement/interaction. Nuclear shape alterations and membrane rearrangements. Nutrient deprivation-induced conditions. Autophagosome formation. NPC clustering/enrichment. Receptor-mediated by Nup159, Atg39 and/or unknown receptor. Autophagic machinery requirement/interaction. Requirement/involvement of auxiliary membrane scission proteins/complexes. Nuclear shape alterations and membrane rearrangements. Nutrient deprivation-induced conditions. Autophagosome formation. Nup159 aggregation. Autophagic machinery requirement/interaction. Nuclear shape alterations and membrane rearrangements.
MODE 2: CLASSICAL MICRONUCLEOPHAGY (yeast Saccharomyces cerevisiae): 1. Piecemeal microautophagy of the nucleus (PMN) [4,8,9,25,26] 2. Atg39-independent micronucleophagy [27,28]	NE components, granular nucleolus, pre-ribosomes, Nop1, Nvj1 Exclusion: NPCs and chromosomal DNA Nucleolar proteins, the CLIP complex (chromosome linkage INM protein complex composed of Src1/Heh1-Nur1) Exclusion: rDNA and rDNAstability-regulating cohibin complex (composed of Csm1-Lrs4)	Nutrient-rich induction and/or nutrient deprivation-induced (short periods) conditions and TORC1 inactivation. Formation of nucleus-vacuole (NV) junctions through Nvj1-Vac8 interaction. Formation of five morphologically distinct PMN intermediates/vesicles. Microautophagic membrane formation/requirement. Autophagic machinery-independent process. Lipid homeostasis requirement. Nuclear shape alterations and vacuolar membrane invagination. Nutrient-rich induction and/or nutrient deprivation-induced conditions. Defects in proteins regulating TORC1 inactivation. PMN-like pathway. Atg39-receptor independent. Nuclear shape alterations and vacuolar membrane invagination.
MODE 3: COMPLETE NUCLEOPHAGY Nucleophagy of the entire nucleus 1. Programmed nuclear death (PND) (ciliated protozoan Tetrahymena thermophila) [29,30] 2. Programmed nuclear destruction (PNuD) (yeast Saccharomyces cerevisiae) [31] 3. Mechanistically uncharacterized nucleophagy in the filamentous fungal species Aspergillus oryzae, Magnoporthe oryzae and Fusarium oxysporum [32–35]	Parental macronucleus Uncellularized nuclei The entire nucleus Whole nuclei Regulating the number of nuclei	Occurs during sexual reproduction. Nuclear membrane rearrangements. Alteration to the composition and distribution of lipids and proteins. Atg8-mediated. Nutrient deprivation induced. Occurs during sporulation/gametogenesis. Apoptotic-like DNA fragmentation and disintegration. Vacuolar membrane dissolution. Nutrient deprivation induced. Formation of ring-like autophagosomes encircling whole nuclei. Atg8-mediated Occurs during appressorium development. Formation of small autophagosomes. Atg1- and/or Atg4-dependent. Occurs during vegetative growth and nutrient-deprivation. Autophagosome-like structure formation. Atg8-mediated. Nuclear membrane alterations.
MODE 4: UNCONVENTIONAL NUCLEOPHAGY (yeast Saccharomyces cerevisiae) Late nucleophagy (LN) [10,36–38]	Bulk nucleoplasm	Nutrient-deprivation induced (prolonged periods of nitrogen starvation). Partial requirement of the core autophagic machinery. Altered nuclear morphology. Nvj1-Vac8 independent. Different from classical micronucleophagy.

Macronucleophagy (and its various modes, namely, bulk macronucleophagy, micronuclear macronucleophagy and chromatophagy/chromatinophagy) is a major and the most common mode of nucleophagy that is primarily observed in mammalian cells under diverse stress-induced, pathological and disease-associated conditions (e.g., genotoxicity, cellular senescence, oncogenic insult, and cell cycle perturbation). The nucleophagic cargo/substrates (e.g., NE, nuclear lamina, chromatin) destined for degradation are encapsulated within double-membrane autophagosomes that subsequently deliver the cargo to lysosomes for ultimate degradation and recycling. The entire process is regulated by the core autophagic machinery and is accompanied by alteration in nuclear membrane size, shape, distribution and intricate nucleus-autophagy membrane interactions (Table 1) [3–7,10–19].

Open questions: Whether basal macronucleophagy in mammalian cells occurs at physiological (non-pathological) levels remains to be elucidated. Currently, the molecular mechanisms/pathways underlying macronucleophagy of nuclear components/nuclei in mammalian cells are still elusive and the specific initiators, receptors and mediators have not yet been identified. There are some indications that macronucleophagy deficiency and/or excess is linked to a multitude of diseases; however, limited understanding of the regulatory and molecular mechanisms and a lack of animal models are currently major obstacles for defining macronucleophagy roles in the pathogenesis of human diseases (e.g., neurodegeneration, cancer, aging, laminopathies).

Macronucleophagy modes, namely, Atg39-dependent macronucleophagy, and most recently, NPC-phagy and nucleoporinophagy have only been detected in the baker’s/budding yeast, Saccharomyces cerevisiae, as an alternative route (to “classical micronucleophagy”) for nuclear degradation. Unlike macronucleophagy in mammalian cells, it is mainly receptor-mediated (e.g., Atg39-ONM resident, Nup159, unknown receptor). Furthermore, this process requires the core autophagic machinery and is responsible for the partial degradation of various components of the nucleus (e.g., NE components, nucleolar proteins, NPC components and/or the NPC as a whole) under nutrient-deprivation conditions. The process is accompanied by intricate nuclear shape alterations and nucleus-autophagy membrane rearrangements and interactions (Table 1) [3–7,20–24].

Open questions: It has been shown that Atg39-dependent macronucleophagy in yeast is involved in the degradation of the nuclear fragments/components; however, it is not clear how those nuclear components are selectively sequestered into phagophores. In addition, the exact roles of Atg39 and its auxiliary mediators in the process are still to be determined. NPCs are highly conserved from yeast to mammals [22–24], therefore, it would be equally interesting to establish if the processes of NPC-phagy and nucleoporinophagy occur in eukaryotes other than yeast and, if so, what are their main initiators, molecular players, and mediators/factors conveying selectivity that determine the turnover rate (and degradation fate) of the NPCs and its components (e.g., nucleoporins)?

“Classical micronucleophagy” and its modes, namely, those of piecemeal microautophagy of the nucleus (PMN) and Atg39-independent micronucleophagy, is the major and most common route of nucleophagy that has been described in S. cerevisiae. This process is induced under nutrient-rich conditions and/or short periods of nutrient deprivation and it is characterized by the formation of direct membrane contacts between the nucleus and lytic vacuole (nucleus-vacuole junctions, NVJs). This is a multistep process that does not involve autophagosome formation but does involve nuclear membrane protrusion (e.g., nuclear blebbing) toward the vacuole/vacuolar invagination by forming a nucleus-vacuole intermediate, which is then isolated from the nucleus and, finally, released into the vacuolar lumen for enzymatic degradation. The delivery of diverse and selected (mostly nonessential) nuclear cargo (e.g., NE components, nucleolar proteins) to the vacuole and its final execution/degradation is partially dependent on the core autophagic machinery, but it heavily relies on lipid and protein homeostasis and membrane rearrangements. The process of “classical micronucleophagy” is poorly studied (or not studied at all) in animal models (Table 1) [3–10,25–28].

Open questions: Most recently, it has been shown that PMN also depends on the Atg39 receptor; however, its involvement and actions are predominantly determined by highly distinct cargo composition and diameter/size, presence of the core autophagic machinery, and membrane composition, rearrangements and curvature. Therefore, larger cargo vesicles containing nuclear components are mainly degraded via the PMN mode, whereas, smaller vesicles containing nuclear components are degraded via Atg39-dependent macronucleophagy [3]. Either way, it is important to indicate that some essential nuclear components such as nucleolar rDNA is spared from nucelophagic degradation and kept in the nucleus [27,28]. Therefore, a more comprehensive understanding of nucleophagic cargo selection, coordination and differentiation through diverse micronucleophagic and/or macronucleophagic modes would be necessary.

“Complete nucleophagy” and its modes, namely, programmed nuclear destruction (PNuD), programmed nuclear death (PND) and uncharacterized nucleophagy are the special types of nucleophagy that involve the degradation of the entire nucleus in S. cerevisiae, ciliated protozoan and filamentous fungi, respectively, under nutrient-deprivation conditions and/or during vegetative growth and sexual reproduction. Diverse membrane, lipid and protein alterations facilitate the formation of autophagosome-like vesicles as well as establishing a direct interaction between the nucleus and vacuole influencing the release of digestive enzymes directly into the nucleus and causing ultimate DNA fragmentation and disintegration (Table 1) [3–7,10,29–35].

Open questions: To date, most studies regarding the process of complete nucleophagy have been performed in multinucleate cells of filamentous fungi and it was found that nucleophagy of the entire nucleus does not cause cell lethality; rather, this process provides nutrients under stressful conditions [29–35]. The identification of relevant molecular players and mechanisms for recognition of nuclei would lead to uncovering how one particular nucleus (from multiple nuclei) is selected and subsequently degraded via nucleophagy. Additional molecular, morphological and biological investigations would be essential in determining any possible relevance of this process in the context of cellular physiology and pathology.

Some unconventional modes of nucleophagy are certain to exist. For example, when yeast cells undergo a prolonged period of starvation (after 24 h), a nucleophagic process such as late nucleophagy (LN), its machinery and membrane alterations would likely be different from both macronucleophagy and classical micronucleophagy, spatially and temporally. LN, a process unlike PMN, does not require the formation of nucleus-vacuole intermediates, indicating a major mechanistic difference between those two nucleophagy modes; however, as with PMN, LN utilizes some of the core autophagic machinery. Distinctively, the absence of a few essential/core autophagy genes hinders LN, which in turn leads to altered/deformed nuclei, implying excessive accumulation of nuclear components that fail to be degraded [3–7,10,36–38].

Open questions: As LN leads to abnormal/altered nuclear structure, it is implied that LN plays a crucial role in the maintenance of nuclear homeostasis [10,36–38]. In the near future, it would be interesting to fully determine the identity and manner by which nuclear material enters the yeast vacuole. Also, it would be interesting to determine how different components of the nucleoplasm are degraded by LN. Moreover, future research should be directed toward elucidating the possible existence and nature of the nucleus-vacuole contact during LN, particularly in the absence of nucleus-vacuole intermediates as well as any mechanistic and/or functional inter-relationship of LN and other nucleophagy modes described in yeast cells.

To conclude, it is obvious that nucleophagy has emerged as a captivating research topic, yet many of its aspects remain to be elucidated [3–39]. While it is a selective type of autophagy, many comparisons with other types of selective autophagy [40–43] have limited relevance for the process due to the fact that nucleophagy, in most instances, selectively targets only a small/selected portion of the nucleus (also known as nibbling of the nucleus) [36–39] at a time, adding an additional level of complexity, intricacy and fine-tuning to its selective modality. To date, collated evidence implies strong nucleophagic influence on intranuclear quality control, cellular homeostasis, physiology, and disease as well as its utilization toward uncovering novel approaches to potential therapeutic treatments; however, this topic still requires supplemental and inspiring research for meaningful outcomes [3–39].

Funding Statement

This work was supported by the National Institute of General Medical Sciences [GM131919].

Disclosure statement

No potential conflict of interest was reported by the authors.