Abstract

   This year marks the 20th anniversary of the discovery that the
   nucleolus can temporarily immobilize proteins, a process known as
   nucleolar sequestration. This review reflects on the progress made to
   understand the physiological roles of nucleolar sequestration and the
   mechanisms involved in the immobilization of proteins. We discuss how
   protein immobilization can occur through a highly choreographed
   amyloidogenic program that converts the nucleolus into a large fibrous
   organelle with amyloid-like characteristics called the amyloid body
   (A-body). We propose a working model of A-body biogenesis that includes
   a role for low-complexity ribosomal intergenic spacer RNA (rIGSRNA) and
   a discrete peptide sequence, the amyloid-converting motif (ACM), found
   in many proteins that undergo immobilization. Amyloid bodies provide a
   unique model to study the multistep assembly of a membraneless
   compartment and may provide alternative insights into the pathological
   amyloidogenesis involved in neurological disorders.

   Keywords: heat shock (HS), acidosis, architectural RNA (arcRNA),
   Alzheimer’s disease, cellular dormancy, physiological amyloidogenesis,
   beta-amyloid protein

Nucleolar Sequestration: Visitors to the Nucleolus

   The role of the nucleolus as the site of ribosome biosynthesis has been
   established since the mid-1960s ([29]Perry, 1960; [30]Perry, 1962;
   [31]Miller and Beatty, 1969). Nucleoli are built around tandem repeats
   of ribosomal DNA (rDNA) in nucleolar organizing regions (NORs) and
   structurally dependent on active transcription of rDNA
   ([32]Hernandez-Verdun, 2006; [33]Raska et al., 2006). Each nucleolus
   consists of a tripartite organization which is classically defined by
   their different appearances under electron microscopy (EM)
   ([34]Pederson, 2011): the fibrillar center (FC), where the RNA
   polymerase I machinery is active; the dense fibrillar component (DFC)
   that is enriched in fibrillarin (FIB1); and the granular component (GC)
   that harbors B23. RNA polymerase I activity is believed to occur at the
   interface between the FC and the DFC, while processing of newly
   synthesized ribosomal RNA (rRNA) and assembly with ribosomal proteins
   occur within the GC ([35]Scheer and Hock, 1999; [36]Thiry and
   Lafontaine, 2005). The traditional role of the nucleolus as a hub of
   rRNA synthesis and ribosome assembly has been the subject of many
   excellent literature surveys ([37]Boisvert, 2007; [38]Pederson, 2011;
   [39]Nemeth and Grummt, 2018). This review focuses on a lesser known
   phenomenon originally coined “nucleolar sequestration,” which describes
   the ability of the nucleolus to sequester regulatory proteins in
   response to cellular cues ([40]Emmott and Hiscox, 2009; [41]Pederson
   and Tsai, 2009; [42]Boulon, 2010).

   2019 marks 20 years since nucleolar sequestration was first
   hypothesized ([43]Bachant and Elledge, 1999) following the discoveries
   that cell cycle regulator Cdc14 phosphatase and E3 ubiquitin ligase
   MDM2 could be temporarily localized in nucleoli to affect cell cycle
   progression ([44]Shou et al., 1999; [45]Visintin et al., 1999;
   [46]Weber, 1999; [47]Lohrum, 2000; [48]Bernardi et al., 2004). In
   Saccharomyces cerevisiae, Cdc14 is sequestered in the nucleolus by
   Cfi1/Net1 until anaphase onset when it is released to promote exit from
   mitosis ([49]Shou et al., 1999; [50]Visintin et al., 1999). In
   mammalian cells, nucleolar sequestration of MDM2 prevents it from
   binding and exporting p53 into the cytoplasm for degradation
   ([51]Weber, 1999; [52]Lohrum, 2000; [53]Bernardi et al., 2004). This
   stabilizes p53 in the nucleus, where it acts as a transcription factor
   that promotes growth arrest or apoptosis. The E3 ubiquitin ligase von
   Hippel–Lindau protein (VHL) is another example of nucleolar
   sequestration ([54]Mekhail, 2004). VHL promotes the degradation of the
   transcription factor hypoxia-inducible factor (HIF) under normal oxygen
   conditions. Low extracellular pH triggers nucleolar sequestration of
   VHL, enabling HIF to evade degradation and promote transcription of
   target genes involved in oxygen homeostasis. [55]Table 1 summarizes the
   reports of nucleolar sequestration that have been observed for many
   other proteins under various cellular conditions. Taken together, these
   underscore a fundamental cellular strategy that uses the nucleolus to
   regulate protein dynamics in response to cellular cues.

Table 1.

   List of the proteins whose activities have been reported to be
   regulated by nucleolar sequestration.
   Protein symbol Full name Stimulus Nucleolar response Model system
   Reference
   Cdc14 Cell division cycle 14 Anaphase Release S. cerevisiae ([56]Shou,
   1999)
   ([57]Visintin et al., 1999)
   Pch2 Pachytene checkpoint 2 Meiotic prophase arrest Release S.
   cerevisiae ([58]San-Segundo and Roeder, 1999)
   MDM2 Murine double minute 2 homolog Ribosomal stress
   DNA damage Capture Mammalian ([59]Weber, 1999)
   ([60]Lohrum, 2000)
   ([61]Bernardi, 2004)
   hTERT Human telomerase reverse transcriptase Transformation, DNA damage
   Ionizing radiation Release
   Capture Mammalian ([62]Wong et al., 2002)
   c-Myc Proto-oncogene c-Myc Proteasomal stress Capture Mammalian
   ([63]Arabi, 2003)
   ADAR2 Adenosine deaminase that acts on RNA 2 Inhibition of rRNA
   synthesis Release Mammalian ([64]Sansam et al., 2003)
   VHL von Hippel Lindau tumor suppressor Extracellular acidosis Capture
   Mammalian ([65]Mekhail, 2004)
   RelA p65 subunit of transcription factor
   NF-κB Aspirin, serum withdrawal, UV-C radiation Capture Mammalian
   ([66]Stark and Dunlop, 2005)
   ([67]Chen and Stark, 2017)
   Polycomb Polycomb Cell differentiation Capture D. melanogaster
   ([68]Chen, 2005)
   Hand1 Heart and neural crest derivatives expressed 1 Cell
   differentiation Capture/Release Mammalian ([69]Martindill et al., 2007)
   Hsc70 Heat shock chaperone 70 Recovery from heat shock Capture
   Mammalian ([70]Banski, 2010)
   Ulp1 Small ubiquitin-related modifier (SUMO) protease Alcohol Capture
   S. cerevisiae ([71]Sydorskyy et al., 2010)
   p53 Cell cycle regulator; tumor suppressor Proteasomal inhibition
   (MG132) Capture Mammalian ([72]Kruger and Scheer, 2010)
   ([73]Latonen, 2011)
   Piwi piRNA binding protein Heat shock Capture D. melanogaster
   ([74]Mikhaleva, 2018)
   [75]Open in a new tab

Nucleolar Sequestration: A Case of Protein Immobilization

The Nucleolar Detention Centers

   Pioneering work by [76]Phair and Misteli (2000) showed that proteins
   are highly mobile and rapidly exchange between affinity interactions
   and the cellular milieu. The nucleolus is considered a dynamic droplet,
   assembled by demixing of its three sub-structural liquid phases (i.e.,
   FC, DFC, and GC), which are composed of highly mobile proteins
   ([77]Brangwynne et al., 2011; [78]Feric et al., 2016; [79]Hult et al.,
   2017; [80]Sawyer et al., 2019). It is remarkable, then, that proteins
   undergoing nucleolar sequestration are non-dynamic or immobile
   ([81]Mekhail, 2004; [82]Mekhail, 2005; [83]Audas et al., 2012;
   [84]Jacob, 2013). This has been demonstrated for Cdc14 ([85]Tomson et
   al., 2009), MDM2 ([86]Audas et al., 2012), VHL ([87]Mekhail, 2005),
   RNF8 ([88]Mekhail, 2007), DNMT1 ([89]Audas et al., 2012), and Piwi
   ([90]Mikhaleva, 2018), amongst other proteins. Even resident nucleolar
   proteins such as RNA polymerase I subunit RPA16, Pescadillo, and SENP3
   can undergo cycles of mobility/immobility ([91]Jacob, 2013). For
   example, VHL switches from a highly dynamic, uniform distribution under
   standard growth conditions (21% O[2], pH 7.4) to an immobilized state
   in the nucleolus on exposure to extracellular acidosis (1% O[2], pH
   6.0) ([92]Mekhail, 2005). Only upon neutralization of extracellular pH
   is VHL released from the nucleolus to return to its original
   distribution and mobility ([93]Mekhail, 2005). These nuclear foci that
   contain sequestered/immobile proteins were originally called “nucleolar
   detention centers,” as targets are both localized and detained within
   the nucleolus, unable to freely diffuse elsewhere ([94]Mekhail, 2005;
   [95]Audas et al., 2012b; [96]Jacob, 2013). From this perspective, the
   function of nucleolar detention or immobilization is to temporarily
   inactivate relevant proteins, inhibiting their access to downstream
   effectors. Just as possible, though, is that the clustering of detained
   proteins may render an enzymatic reaction more efficient. For example,
   immobilized nucleolar Cdc14 maintains Spo12 dephosphorylation to
   regulate cell cycle progression ([97]Tomson et al., 2009). In addition,
   Piwi switches from its canonical role as a non-nucleolar transposable
   element repressor to a rDNA-specific repressor when it is sequestered
   in the nucleolus ([98]Mikhaleva, 2018). From these studies, it is clear
   that cells have evolved a strategy to regulate molecular networks by
   reversibly switching proteins between a mobile and an immobile state.
   Whether nucleolar sequestration represents a loss- or gain-of-function
   might depend on the proteins undergoing immobilization.

Mechanisms of Nucleolar Sequestration

   The ability of the nucleolus to sequester a wide variety of proteins in
   various cellular contexts suggests multiple mechanisms for nucleolar
   sequestration. Pathway analysis of interactions between resident
   nucleolar proteins and visitor proteins indicates that sequestered
   proteins interact either directly or indirectly, with the same small
   subset of “hub” nucleolar proteins, which primarily includes B23/NPM
   and Nucleolin ([99]Emmott and Hiscox, 2009). Nucleolar retention of
   highly dynamic proteins through interactions with less mobile “hub”
   nucleolar partner(s) anchored by multivalent protein and RNA
   interactions contribute to nucleolar plasticity ([100]Mitrea et al.,
   2016). Cdc14 is anchored in the nucleolus for most of the cell cycle
   through its association with Cfi1/Net1 ([101]Shou et al., 1999;
   [102]Visintin et al., 1999). During anaphase onset, a signaling cascade
   of phosphorylation inhibits the interaction of Cdc14 with Net1,
   releasing it to act as a mitotic exit activator ([103]Azzam et al.,
   2004). ARF binding to MDM2 unmasks a cryptic nucleolar localization
   signal (NoLS) within its C-terminal RING domain, which is essential for
   MDM2 nucleolar sequestration ([104]Tao and Levine, 1999; [105]Weber,
   1999; [106]Lohrum, 2000; [107]Weber et al., 2000). During DNA damage
   and acidosis, MDM2 is shuttled into the nucleolus by direct binding to
   PML (promyelocytic leukemia protein) ([108]Bernardi et al., 2004),
   itself a target of nucleolar sequestration ([109]Mattsson, 2001). MDM2
   can also be sequestered in nucleoli by binding to ATP, independently of
   ARF ([110]Poyurovsky et al., 2003). Alternatively, MDM2 binds ribosomal
   proteins (e.g., RPL5 and RPL11) that are released into the nucleoplasm
   during ribosomal stress, which stabilizes p53 ([111]Boulon, 2010;
   [112]Liu et al., 2016). Mapping analysis of VHL identified an
   approximately 30-amino acid fragment referred to as a nucleolar
   detention signal (NoDS) that is necessary and sufficient to immobilize
   proteins in nucleoli ([113]Mekhail, 2007). The NoDS is composed of an
   arginine/histidine-rich sequence followed by two or more hydrophobic
   LXV motifs where X can be any hydrophobic residue (e.g., LWL, LLV, LFV,
   and LQV). A survey of proteins containing NoDS identified many
   candidates harboring this motif, including DNA methyltransferase I
   DNMT1, PML, and DNA polymerase delta catalytic subunit POLD1, all shown
   to be immobilized in nucleoli under extracellular acidosis
   ([114]Mekhail, 2007; [115]Audas et al., 2012a). The NoDS interacts with
   inducible long noncoding RNA derived from the ribosomal intergenic
   spacer (rIGSRNA) ([116]Audas et al., 2012a). Silencing of rIGSRNA is
   sufficient to prevent both the formation of nucleolar detention centers
   and immobilization of NoDS-containing proteins ([117]Audas et al.,
   2012a). The identification of the NoDS in many proteins provided
   evidence that nucleolar sequestration is a common cellular strategy to
   regulate protein function ([118]Mekhail, 2007).

Biochemical Properties of Nucleolar Detention Centers

Nucleolar Detention Centers Display Amyloidogenic-Like Characteristics

   In various physiological settings, proteins with limited mobility often
   display amyloid-like properties ([119]Kayatekin et al., 2014;
   [120]Berchowitz et al., 2015). Amyloidogenesis is the process whereby
   soluble proteins assemble into aggregates known as amyloid fibrils
   ([121]Knowles et al., 2014). Because of their association to
   neurodegenerative disorders, including Alzheimer’s and Parkinson’s
   disease, amyloids were classically perceived as exclusively toxic
   protein aggregates ([122]Schnabel, 2010; [123]Knowles et al., 2014).
   The discovery of functional amyloid Pmel17 in 2006 challenged the
   notion that the amyloid state is merely pathological ([124]Fowler,
   2006). Since then, several groups have reported the existence of
   functional/physiological amyloids in different organisms ([125]Chiti
   and Dobson, 2006; [126]Fowler, 2007; [127]Maji et al., 2009;
   [128]Falabella et al., 2012; [129]Fowler and Kelly, 2012; [130]Li et
   al., 2012; [131]Kayatekin et al., 2014; [132]Berchowitz et al., 2015;
   [133]Tang et al., 2015; [134]Saad, 2017; [135]Cereghetti, 2018).
   Nucleolar detention centers composed of proteins such as VHL, MDM2,
   POLD1, etc., share many properties beyond immobility that are
   associated with the amyloid state. First, these nuclear foci stained
   positive with amyloidophilic dyes such as Congo red, Thioflavin S/T,
   and Amylo-Glo, all of which recognize different biochemical features of
   amyloids ([136]Audas et al., 2016). Second, the nucleolar detention
   centers stain positive for the OC fibril antibody that specifically
   targets the amyloid fibril conformation ([137]Kayed et al., 2007).
   Third, immobilized proteins found in these nuclear foci displayed
   biochemical properties associated with amyloids, including resistance
   to proteinase K, insolubility in common detergents, and can only be
   dissociated into monomers by SDS/high temperature ([138]Audas et al.,
   2016). Arguably the utmost unique feature of amyloid bodies is their
   electron-dense fibrillar organization, which would be as predicted for
   a condensate enriched in amylogenic proteins ([139]Jacob, 2013;
   [140]Audas et al., 2016). Consequently, the terms “nucleolar detention
   centers” and NoDS were replaced with “amyloid bodies” (A-bodies) and
   “amyloid-converting motif” (ACM), respectively, to reflect the
   transformation of the nucleolus into an A-body, a molecular prison of
   proteins in their amyloid-like state. [141]Figure 1A shows an example
   of this dramatic and reversible transformation of the tripartite
   nucleolus into the fibrillar A-body in cells responding to stimuli.

Figure 1.

   Figure 1
   [142]Open in a new tab

   Nucleolar sequestration: the reversible remodeling of the nucleolus
   into an amyloid body. (A) During stimuli (heat shock or extracellular
   acidosis), the tripartite nucleolus undergoes a dramatic transformation
   into electron-dense fibrillar organization that characterizes an
   amyloid body. The fibers contain immobilized proteins in an
   amyloid-like state. After stimuli termination, an amyloid body is
   disaggregated and transforms back into the tripartite nucleolus. (B)
   The fibrillar amyloid bodies are distinct from the amorphous,
   electron-dense nucleolar caps (16 h cisplatin) or the electron-light
   nucleolar aggresomes (16 h MG132). FC, fibrillar component; DFC, dense
   fibrillar component; GC, granular component. Scale represents 1 µm.
   Amyloid body and nucleolar aggresome taken from ([143]Audas, 2016) and
   ([144]Kruger and Scheer, 2010), with permission.

Amyloid Bodies Are Distinct From Liquid-Like Biomolecular Condensates and
Other Nucleolar Organizations

   Purification and mass spectrometry analysis have identified hundreds of
   cellular proteins that are captured in A-bodies, many of which were
   shown to undergo immobilization by photobleaching experiments
   ([145]Mekhail, 2007; [146]Audas et al., 2012a; [147]Audas et al.,
   2016). Because A-bodies contain an array of immobilized proteins in an
   amyloid-like state, we suggested the term “systemic physiological
   amyloidogenesis” to describe A-body biogenesis, in keeping with the
   original terminology to describe functional amyloids ([148]Chiti and
   Dobson, 2006; [149]Fowler, 2007; [150]Maji et al., 2009). Other
   laboratories have also proposed that A-body formation represents an
   amyloidogenic liquid-to-solid phase transition ([151]Lyons and
   Anderson, 2016; [152]Latonen, 2019; [153]Hall et al., 2019) and used
   the terms “solid,” “solid-like,” or “non-dynamic” biomolecular
   condensates to describe this membraneless organelle ([154]Lyons and
   Anderson, 2016; [155]Weber, 2017; [156]Cereghetti, 2018; [157]Holehouse
   and Pappu, 2018; [158]Woodruff et al., 2018; [159]Itakura et al., 2018;
   [160]Fay and Anderson, 2018; [161]Latonen, 2019). Prior to the
   discovery of A-bodies, the terms “liquid-to-solid phase transition” and
   “solid” had been reserved to describe the formation of pathological
   aggregates ([162]Patel et al., 2015; [163]Murakami et al., 2015;
   [164]Jain and Vale, 2017; [165]Mateju et al., 2017; [166]Peskett, 2018;
   [167]Posey et al., 2018). Other physiological examples of “solid-like”
   structures include Balbiani bodies observed in Xenopus ([168]Boke et
   al, 2016) and pH-regulated fluid-to-solid transition of the cytoplasm
   in yeast ([169]Munder et al, 2016). The fibrous, amyloid-like
   characteristic of the A-body differentiates it from other biomolecular
   condensates that display liquid-like properties ([170]Brangwynne et
   al., 2009; [171]Brangwynne et al., 2011; [172]Weber and Brangwynne,
   2012; [173]Zhu and Brangwynne, 2015; [174]Banani, 2017; [175]Shin and
   Brangwynne, 2017), such as stress granules, nucleoli, and paraspeckles,
   amongst others ([176]Hodges, 1998; [177]Gall, 2000; [178]Lamond and
   Spector, 2003; [179]Rizzi et al., 2004; [180]Dellaire and Bazett-Jones,
   2004; [181]Valgardsdottir, 2005; [182]Parker and Sheth, 2007;
   [183]Sasaki and Hirose, 2009; [184]Bond and Fox, 2009; [185]Machyna et
   al., 2013; [186]Pederson, 2011; [187]Protter and Parker, 2016).
   Liquid-like biomolecular condensates are dynamic, their constituents
   are mobile, they do not form fibers detectable by EM nor do they
   typically stain with amyloidophilic dyes ([188]Phair and Misteli, 2000;
   [189]Shin and Brangwynne, 2017; [190]Weber, 2017). The biochemical and
   biophysical differences between dynamic, liquid-like, and non-dynamic
   or solid-like condensates are summarized within [191]Table 2 .

Table 2.

   Biochemical, biophysical, and dynamic properties of liquid-like
   condensates or solid-like condensates with amyloid characteristics.
   Liquid-like condensates Solid-like condensates
   Examples Cytoplasm stress granules ([192]Protter and Parker, 2016)
   P-bodies ([193]Parker and Sheth, 2007)
   Nuclear stress granules ([194]Rizzi et al., 2004; [195]Valgardsdottir
   et al., 2005)
   Cajal bodies ([196]Gall, 2000; [197]Machyna et al., 2013)
   Nuclear speckles ([198]Lamond and Spector, 2003)
   Nuclear paraspeckles ([199]Bond and Fox, 2009; [200]Sasaki and Hirose,
   2009)
   Nucleoli ([201]Pederson, 2011)
   PML nuclear bodies ([202]Hodges et al., 1998; [203]Dellaire and
   Bazett-Jones, 2004) Amyloid bodies ([204]Audas, 2016)
   Balbiani bodies ([205]Boke, 2016)
   Protein mobility Proteins are mobile; continuously exchanging with the
   structure and the surrounding milieu Proteins are immobile; engaged in
   strong intermolecular interactions
   Shape Spherical Spherical or fibrous
   Biochemical and biophysical characteristics • Structure is dynamic;
   exhibiting properties of water droplets:Fluid
   • Cycles of fusion (coalescence) and fission
   • Wetting behavior
   • Flows under shear force • Structure is non-dynamic; exhibiting
   properties of amyloids:Static
   • Fibrillar organization
   • Positive staining with amyloidophilic dyes (e.g., Congo red)
   • Resistant to proteinase K
   • Insoluble in common detergents
   • Cross-β diffraction pattern
   Material properties Viscous Elastic
   Function Biochemical reactions Cell dormancy
   Mechanism Liquid–liquid phase separation Liquid-to-solid phase
   transition
   [206]Open in a new tab

   A-bodies may also be contrasted from other stress-induced nucleolar
   structures, namely, nucleolar caps ([207]Shav-Tal et al., 2005) and
   nucleolar aggresomes ([208]Latonen, 2011; [209]Latonen, 2011;
   [210]Latonen, 2019) ([211] Figure 1B ). Transcriptional inhibition of
   Pol I results in nucleolar segregation, in which the FC and GC phases
   separate, perhaps as a consequence of changes in surface tension
   ([212]Shin and Brangwynne, 2017) resulting in the formation of
   perinucleolar caps that surround the segregated nucleolus
   ([213]Shav-Tal et al., 2005). Proteasomal inhibition induces nucleolar
   inclusions called nucleolar aggresomes that contain proteins marked for
   degradation, and various RNAs ([214]Latonen, 2011). By EM, A-bodies
   have a unique fibrillar organization characteristic of amyloids,
   whereas nucleolar caps appear as electron-dense amorphous structures
   and nucleolar aggresomes are cavernous, occupying a large
   electron-light central space of the nucleolus ([215]Kruger and Scheer,
   2010) ([216] Figure 1B ). Interestingly, while the formation of
   nucleolar caps and A-bodies is accompanied by a redistribution of
   nucleolar components and subsequent arrest of ribosomal biogenesis
   ([217]Shav-Tal et al., 2005; [218]Mekhail, 2006; [219]Jacob, 2013),
   nucleolar aggresomes require transcriptionally active nucleoli to form
   ([220]Latonen, 2011; [221]Latonen, 2011), with all nucleolar components
   intact and visible by EM. Additionally, proteins are dynamically sorted
   into nucleolar caps and remain mobile ([222]Shav-Tal et al., 2005),
   while several proteins in A-bodies and nucleolar aggresomes are
   immobile ([223]Mekhail, 2005; [224]Audas et al., 2012a; [225]Jacob,
   2013; [226]Audas et al., 2016). Another recently identified nucleolar
   stress body, nucleolar amyloid bodies (NoABs), is induced by
   prematurely terminated peptides that diffuse through the nuclear pores
   and aggregate within the nucleolus ([227]Mediani et al., 2019). EM has
   not been reported for NoABs. It remains to be tested if NoABs are
   rIGSRNA-dependent, as are A-bodies, and if prematurely terminated
   peptides are involved in nucleolar sequestration of full-length
   proteins. While there are definitive similarities between the different
   stress-induced nucleolar organizations and that proteins can undergo
   sequestration in nucleolar aggresomes (both are discussed in
   ([228]Latonen, 2019), A-bodies represent a unique nucleolar structure
   based on their fibrous properties and dependency on rIGSRNA.

Protein Immobilization Into A-Bodies: A Choreographed Multistep Pathway

   How membraneless subcellular condensates maintain their unique
   identities and how proteins and/or RNA are sorted into these
   condensates remain subjects of active research. New insights into the
   biophysical properties of biomolecular condensates have demonstrated
   unexpected links between the sequence-encoded information in protein
   and RNA components of compartments and the material properties they
   impart ([229]Kato et al., 2012; [230]Altmeyer et al., 2015;
   [231]Brangwynne et al., 2015; [232]Elbaum-Garfinkle et al., 2015;
   [233]Molliex et al., 2015; [234]Nott et al., 2015; [235]Pak et al.,
   2016; [236]Shin and Brangwynne, 2017; [237]Weber, 2017; [238]Langdon,
   2018). Recent studies demonstrated the importance of RNA-binding
   proteins with prion-like, low-complexity domains in forming
   biomolecular condensates. Equally as exciting has been the work done to
   uncover the specific sequences or structural elements embedded in
   architectural RNA that dictate the biophysical properties of
   biomolecular condensates ([239]Chujo, 2017; [240]Chujo and Hirose,
   2017; [241]Yamazaki et al., 2018; [242]Hirose et al., 2019). With these
   new tools, we have begun to understand how specific elements within RNA
   and proteins are involved in condensate biogenesis.

A Working Model of A-Body Biogenesis

   [243]Figure 2 proposes a stepwise working model of A-body biogenesis
   that integrates different opinions in the literature, showcasing this
   process as a precisely choreographed multistep routine rather than
   simply a random aggregation of misfolded proteins. The first step in
   the formation of A-bodies is the appearance of several transient foci
   that are distinct from known nucleolar layers ([244]Wang et al., 2018).
   These foci are spherical, contain mobile proteins, and can undergo
   fusion, thereby displaying some of the properties associated with
   dynamic condensates. An interesting feature of the inducible nucleolar
   foci is that they stain positive for 1-anilino-8-naphthalenesulfonate
   (ANS), a dye specific for hydrophobic regions of proteins and used in
   vitro to detect the molten globule state, a precursor of amyloid
   fibrils ([245]Booth, 1997). EM revealed that ANS-positive foci
   correspond to electron-dense, amorphous aggregates concomitant with
   loss of the typical tripartite organization of transcriptionally active
   nucleoli. In the second step, the stimulus-induced foci rapidly mature
   into Congo red-positive aggregates that contain immobilized proteins,
   limiting their ability to diffuse within the nucleolus. We coined these
   maturing foci “nascent A-bodies.” Photobleaching analysis showed that,
   once formed, nascent A-bodies expand by directly capturing and
   immobilizing free proteins (step 3) ([246]Wang et al, 2018). Maturation
   of A-bodies terminates once the pools of cellular mobile proteins have
   been depleted, culminating in a distinct fibrous organization.
   Disassembly of A-bodies occurs within 1–2 h after stimulus termination,
   a process that requires heat shock proteins hsp70 and hsp90 (step 4)
   ([247]Audas et al., 2016).

Figure 2.

   [248]Figure 2
   [249]Open in a new tab

   Working model: amyloid body biogenesis is a precisely choreographed
   routine. We propose that, on stimulus, low-complexity ribosomal
   intergenic spacer RNA (rIGSRNA) derived from the rDNA intergenic spacer
   accumulate in the nucleolus. Step 1: Low-complexity rIGSRNA interact
   with short cationic peptides, such as the R/H-rich sequence of the ACM
   (formally NoDS), to form nucleolar liquid-like foci. Step 2: Local
   concentration of proteins with amyloidogenic propensity in the foci
   triggers physiological amyloidogenesis and generates nascent amyloid
   bodies (A-bodies). Step 3: Once seeded, nascent A-bodies self-assemble
   into fibrillar, solid-like A-bodies. A-bodies enable cells to rapidly
   and reversibly store a large array of proteins and enter cellular
   dormancy in response to stress. Step 4: Upon recovery/stimulus
   termination, A-body disaggregation is mediated by heat shock protein
   (hsp) chaperones 70 and 90. Through these steps, A-body biogenesis may
   represent a physiological liquid-to-solid phase transition.

Low-Complexity rIGSRNA Drive Formation of Inducible Nucleolar Foci

   In mammals, the nucleolus is organized around a scaffold of ∼400 rDNA
   tandem repeats of 43 kb, of which approximately half are
   transcriptionally active ([250]Nemeth and Grummt, 2018; [251]Sharifi
   and Bierhoff, 2018). Each repeat consists of an rDNA enhancer/promoter
   located directly upstream of rRNA genes separated by a ribosomal
   intergenic spacer (rIGS) of variable length and organization
   ([252]Gonzalez and Sylvester, 1995; [253]Smirnov, 2016) ([254] Figure
   3A ). The rIGS is an enigmatic region of the human genome historically,
   and erroneously, called the “non-transcribed region” ([255]Smirnov,
   2016). Interestingly, in recent years, species conservation
   ([256]Agrawal and Ganley, 2018) and functional studies have
   demonstrated that these regions of the genome are transcriptionally
   active, generating various non-coding RNA (ncRNA) ([257]Jacob, 2012;
   [258]Audas and Lee, 2016). These ncRNA from the rIGS are involved in
   regulating rRNA expression ([259]Mayer, 2006; [260]Mayer et al., 2008;
   [261]Schmitz, 2010; [262]Zhao, 2016; [263]Zhao, 2016; [264]Zhao, 2018)
   and thereby responsible for maintaining a significant fraction of the
   rDNA cassettes in a heterochromatic, transcriptionally silent chromatin
   structure ([265]Grummt and Pikaard, 2003; [266]Santoro, 2005),
   controlling PTBP1-regulated alternative splicing ([267]Yap, 2018), and
   assembling A-bodies ([268]Audas et al., 2012a; [269]Jacob, 2013;
   [270]Audas et al., 2016; [271]Wang et al., 2018) ([272] Figure 3A ).
   The various rIGS ncRNA appear to be products of RNA polymerase I
   ([273]Mayer, 2006; [274]Audas et al., 2012a), except for the antisense
   PAPAS, which is transcribed by RNA polymerase II ([275]Zhao, 2016).

Figure 3.

   [276]Figure 3
   [277]Open in a new tab

   Induction of non-coding RNA (ncRNA) from the ribosomal cassette. (A)
   Schematic of a single human rDNA repeat unit, which is composed of a
   ∼13-kb pre-rRNA coding region flanked by a ∼30-kb intergenic spacer
   (rIGS). The rIGS transcribes several functional non-coding RNA.
   Stimuli-specific loci of rIGS produce ribosomal intergenic spacer RNA
   (rIGSRNA) required for A-body formation. rIGS[28]RNA and
   rIGS[16]RNA/rIGS[22]RNA are produced under acidotic (yellow) and heat
   shock (red) conditions, respectively. No function has been ascribed to
   rIGS[18]RNA yet. Other ncRNA found in the rIGS include a >10-kb
   transcript called PNCTR (pyrimidine-rich non-coding transcript)
   involved in PTBP1 binding (purple), pRNA (green), and antisense PAPAS
   (brown) involved in rRNA regulation, as well as Alu element-derived
   (gray boxes) RNA involved in nucleolar architecture. (B) rIGSRNA
   contain low-complexity sequences comprising of long dinucleotide
   repeats, as determined by RNA sequencing, RT cloning, and RNA-FISH.
   This is in contrast to other ncRNA that display high complexity, i.e.,
   possess secondary structure, such as pRNA.

   The appearance of nucleolar foci under stress coincides with the
   induction of rIGS[28]RNA in acidosis, and rIGS[16]RNA and rIGS[22]RNA
   in heat shock ([278] Figure 3 ). Recent work suggests that
   low-complexity dinucleotide repetitive sequences operate as the
   architectural determinants of rIGSRNA to recruit proteins to A-bodies
   ([279]Wang et al., 2018) ([280] Figure 3B ). Live cell imaging and in
   vitro assays indicated that positively charged R/H-rich peptide domains
   of the ACM ([281] Figure 4 ) co-assemble more efficiently with the
   negatively charged low-complexity sequences of rIGSRNA than with
   flanking high-complexity RNA sequences to form the inducible nucleolar
   foci ([282]Wang et al., 2018). The rIGSRNA/ACM interactions in vitro
   and in vivo are particularly sensitive to even the slightest increase
   in salt concentration. This contrasts other phase-separated
   compartments of the nucleolus, which are disrupted at considerably
   higher salt concentrations. Thus, the interactions between
   low-complexity RNA and the R/H-rich region of the ACM characteristic of
   step 1 in A-body biogenesis ([283] Figure 2 ) appear to be
   electrostatic in nature and are likely driven by complex coacervation,
   a charge-based form of liquid–liquid phase separation that has been
   observed in other cellular settings in vivo ([284]Altmeyer et al.,
   2015; [285]Pak et al., 2016).

Figure 4.

   Figure 4
   [286]Open in a new tab

   The amyloid-converting motif (ACM). (A) The ACM is necessary and
   sufficient to target and immobilize proteins in amyloid bodies
   (A-bodies). It consists of a R/H-rich short cationic domain flanking a
   high fibrillation propensity domain (determined by Rosetta energy of
   less than −23 kcal/mol). (B) We propose that it is its bipartite nature
   that allows the ACM to traverse phase boundaries. Complex coacervation
   of short cationic domains and low-complexity rIGSRNA (liquid–liquid
   phase separation) concentrates fibrillation propensity domains to
   activate a liquid-to-solid phase transition to form A-bodies.

Amyloidogenic Properties of the ACM Trigger Protein Immobilization

   In principle, A-bodies and Balbiani bodies should be composed of
   proteins that possess fibrillation propensity, i.e., increased
   likelihood of forming fibrils. Bioinformatic analysis of the consensus
   ACM revealed the hydrophobic LXV motifs make up a region of high
   fibrillation propensity, as predicted by a Rosetta energy score of less
   than −23 kcal/mol ([287]Goldschmidt, 2010) ([288] Figure 4A ). As a
   whole, the ACM bears a striking resemblance to that of the prototypical
   pathological β-amyloid, historically associated with Alzheimer’s
   disease, in which a R/H-rich sequence is in close proximity to a region
   of high fibrillation propensity referred to in literature as the P3
   fragment ([289] Figure 4A ) ([290]Dulin et al., 2008). The ACM exhibits
   classic amyloidogenic properties previously observed for β-amyloid,
   namely, the cross-β X-ray diffraction pattern in vivo and the ability
   to form fibrils in vitro ([291]Audas et al., 2016).

   It has been proposed that the function of the initial liquid state is
   to locally concentrate proteins with fibrillation propensity that would
   otherwise be at levels below the critical threshold in the cell
   ([292]Knowles et al., 2014). Concentration-dependent activation of
   protein fibrillation has been well-documented in vitro ([293]Eisenberg
   and Jucker, 2012; [294]Knowles et al., 2014). Indeed, markers of early
   or nascent A-bodies include ANS and A11 staining ([295]Wang et al.,
   2018) that are typically used in vitro to indicate accumulating
   pre-amyloidogenic structures ([296]Booth, 1997; [297]Kayed et al.,
   2007; [298]Hawe et al., 2008; [299]Tang et al., 2015). In addition,
   photobleaching analyses revealed that the core of nucleolar foci
   contain immobile proteins. Nascent A-bodies expand by self-assembly,
   during which soluble proteins are added directly and autonomously to
   the growing amyloid structure ([300] Figure 2 ) ([301]Wang et al.,
   2018).

The ACM Traverses Across Phase Boundaries to Confer A-Body Identity

   As described above, low-complexity RNA sequences are important in
   conferring A-bodies their unique identity ([302]Wang et al., 2018),
   adding to the list of architectural determinants, which includes
   sequence-specific RNA ([303]Yamazaki et al., 2018), mRNA secondary
   structure ([304]Jain and Vale, 2017; [305]Langdon, 2018), and short
   unstructured RNA in vitro ([306]Nott et al., 2016). The low-complexity
   rIGSRNA-mediated foci themselves appear immiscible from the three
   canonical compartments of the nucleolus ([307]Feric et al., 2016;
   [308]Wang et al., 2018), suggesting they exhibit distinct properties
   that may exclude mixing. Ultimately, it is the bipartite nature of the
   ACM that confers A-body identity and differentiates the ACM from other
   motifs. One possibility is that the R/H-rich “short cationic domain”
   mediates complex coacervation while the “fibrillation propensity
   domain” initiates amyloidogenesis to immobilize proteins in A-bodies
   ([309]Audas et al., 2016; [310]Wang et al., 2018) ([311] Figure 4B ).
   In this model, proteins without a region of high fibrillation
   propensity are unlikely to be incorporated into A-bodies. In other
   words, while many proteins containing positively charged domains can
   form transient rIGSRNA-dependent nucleolar foci, only the ones that
   harbor high fibrillation propensity sequences will be found immobilized
   in A-bodies. The balance of positive to negative charge ratio, presence
   of polar and/or aromatic residues, and modifications ([312]Aumiller and
   Keating, 2016; [313]Monahan et a.l, 2017) will affect how ACM engage in
   intermolecular interactions with other proteins and low-complexity RNA.
   Therefore, the ACM is versatile as it can physiologically transition
   proteins across phase boundaries.

Physiological Amyloids Promote Cell Dormancy

   Endogenous A-bodies have been found in primary cultures and cell lines
   exposed to various stimuli, the cores of human tumors, and subsets of
   cells in normal human and mouse tissues ([314]Audas et al., 2016). So,
   while A-bodies share structural features with pathological amyloids,
   their ubiquitous and reversible nature is indicative of a physiological
   function. Pathway enrichment analysis determined that many of the
   proteins sequestered in A-bodies are involved in cell cycle regulation
   and DNA synthesis, such as CDK1, POLD1, and DNMT1 ([315]Audas et al.,
   2016). By temporarily detaining key factors from their sites of
   activity into A-bodies, major molecular networks are disrupted,
   suppressing metabolic activity ([316]Mekhail, 2006) and arresting
   proliferation/DNA synthesis ([317]Audas et al., 2016). This is
   different from the quiescent state (G0) in the cell cycle where cells
   arrest proliferation but remain metabolically active. Therefore, the
   biological role of A-body formation is to promote cellular dormancy as
   an adaptive response to environmental stressors. These findings are
   consistent with reports that show Balbiani bodies ([318]Boke et al.,
   2016) and fluid-to-solid transitions in yeast ([319]Munder et al, 2016)
   promote dormancy, reinforcing the concept that cells utilize different
   states of matter to perform various biological functions ([320] Table 2
   ). The material properties of “solid-like” systems—non-dynamic,
   amyloid-like—result in loss of metabolic activity and promote cellular
   dormancy, while those of ”liquid-like” systems—dynamic,
   fluid-like—concentrate and facilitate biochemical reactions.

A-Bodies as Targets for Therapeutic Discovery

Implications for Pathological Amyloids

   A-bodies that are physiologically produced remain confined within the
   nucleus and are considered non-toxic amyloids ([321]Wang et al., 2017).
   How the nuclear environment alters the interaction properties of
   aggregation-prone proteins to prevent toxicity ([322]Woerner et al.,
   2016; [323]Maharana, 2018) compared to the cytoplasm is unclear. These
   results imply the cell tightly regulates the induction and degradation
   of low-complexity rIGSRNA within the nucleolus in response to stimuli.
   It is interesting that the pathological β-amyloid involved in
   Alzheimer’s disease is an ACM and undergoes immobilization in A-bodies.
   Mutations that decrease the fibrillation potential of β-amyloid prevent
   its immobilization in A-bodies, providing a link between β-amyloid
   fibrillation and A-body biogenesis. It is tempting to speculate that
   disruptions of A-body biogenesis, for example by cell death, may
   provide the initial seeds for pathological amyloidogenesis. Whether
   aberrant production or localization of low-complexity RNA or exposure
   of A-bodies to the extracellular environment is involved in amyloid
   pathologies such as Alzheimer’s and Huntington’s diseases will be the
   subject of future studies.

Implications for Tumor Cell Dormancy and Metastasis

   Given the evidence that A-bodies are observed in the cores of tumors
   and promote cell dormancy ([324]Audas et al., 2016), perhaps there is a
   link between A-body formation and tumor cell dormancy. This could allow
   cancer cells to adapt to the harsh hypoxic/acidotic conditions of the
   tumor microenvironment. Such a connection has pertinent implications
   for chemotherapeutic resistance and metastasis ([325]Li, 2014; [326]Li,
   2015). With major metabolic pathways shut down and key drug targets
   stored away in A-bodies, a small population of dormant tumor cells may
   be resistant to treatment, survive for a prolonged period of time, and
   contribute to disease recurrence or metastasis upon their metabolic
   reactivation. Preventing cancer cells from forming A-bodies and going
   dormant or, more realistically, preventing cancer cells from
   reactivating may become a viable treatment option.

   An interesting avenue of research that may offer a more effective
   cancer treatment strategy involves manipulating the proteins that evade
   capture into A-bodies. Interestingly, proteins that remain active to
   sustain basal metabolism and viability under stress tend to be devoid
   of fibrillation propensity domains and evade capture into A-bodies.
   These proteins would be more susceptible to manipulation and serve as
   better chemotherapeutic targets than proteins captured in A-bodies.

Discussion

   Twenty years after its discovery, the study of nucleolar sequestration
   has led to important conceptual and mechanistical advances in our
   understanding of the role of membraneless bodies and how they are
   constructed. The ability of cells to reversibly cycle proteins from a
   mobile to immobile state in A-bodies represents an effective
   posttranslational mechanism to regulate molecular networks. In this
   review, we propose a stepwise working model of A-body biogenesis that
   highlights this process as a precisely choreographed multistep routine
   rather than a random aggregation of misfolded proteins. This also makes
   A-bodies unique from the liquid compartments that populate the cell.
   A-bodies are characterized by electron-dense fibers composed of an
   array of immobilized proteins that stain positive with various
   amyloidogenic dyes. Liquid condensates do not display amyloidogenic
   features and generally contain mobile proteins. Obviously, this raises
   the key question of why liquid condensates typically do not mature into
   bodies with amyloidogenic properties. One possibility is that the
   clusters of low-complexity rIGSRNA are able to recruit sufficient
   quantity of proteins by simple electrostatic interactions, thereby
   reaching the critical concentration threshold to trigger
   amyloidogenesis ([327]Knowles et al., 2014). This model is supported by
   the observation of electron-dense material early on in A-body
   biogenesis. The bipartite nature of the ACM, which can both
   electrostatically interact with low-complexity rIGSRNA and contains a
   high fibrillation propensity domain necessary for immobilization, may
   also explain the maturation of A-bodies. Perhaps the architectural
   determinants of RNA that seed various liquid bodies are too restrictive
   to certain binding partners to reach a concentration threshold required
   for amyloidogenesis. Additionally, there is evidence that suggests high
   nuclear RNA concentration acts as a buffer to prevent condensates from
   becoming amyloidogenic ([328]Maharana et al., 2018). Indeed, mutated
   proteins with increased propensity to form amyloids are preferentially
   formed in the cytoplasm, which has low RNA concentration. While this
   appears to be the case for cytoplasmic stress granules enriched in
   amyloidogenic mutant proteins, this likely does not explain A-body
   maturation as the nucleolus is highly enriched in rRNA. How A-bodies
   progress from liquid-like condensates to solid-like condensates is a
   major question in the field of condensate biology, which we are
   actively pursuing. We are also keen to explore other stimuli,
   particularly those that alter nucleolar architecture, that may promote
   the accumulation of rIGSRNA and the formation of A-bodies, including
   viral infection ([329]Wang, 2010), inflammation, and diurnal cycles of
   metabolism. Diurnal oscillations in nucleolar size and abundance of
   nucleolar RNAs have been seen in mammalian systems ([330]Sinturel et
   al., 2017; [331]Aitken and Semple, 2017), as well as an emerging
   connection between circadian disturbances and Alzheimer’s disease
   ([332]Musiek, 2015). Despite most proteins having inherent fibrillation
   propensity, the dominant view remains that amyloids are inherently
   toxic, rather than a physiological fold exploited by cells to regulate
   various peptide functions. The challenge will be to decipher
   fundamental differences between A-bodies and other physiological or
   pathological membraneless compartments that could inform our
   understanding on how to prevent, detect, and treat amyloid-based
   diseases.

Author Contributions

   MW, MB, PT, and SL designed the manuscript, discussed the conceptual
   implications, wrote the paper, and made the figures.

Funding

   This work was supported by grants from the National Institute of
   General Medical Sciences (R01GM115342) (SL), the National Cancer
   Institute (R01CA200676) of the NIH, and the Sylvester Comprehensive
   Cancer Center (SL).

Conflict of Interest

   The authors declare that the research was conducted in the absence of
   any commercial or financial relationships that could be construed as a
   potential conflict of interest.

References