Nucleolus

 

The nucleolus (or nucleoli – sometimes there are more than one) lies at the approximate center of the nucleus. It is not enclosed by a membrane, but is in direct contact with the surrounding nucleus, allowing for the free movement of information that’s essential to its chief function as the central timekeeper of the cell.¹  Together with nucleosomes and the ribosomes it transcribes and assembles, the nucleolus creates the shape-based source of living time that determines the fundamental spacing of succession reflected in all transcription and translation activities of the cell.  If, as some scientists suggest, the nucleus is “the brain of the cell,” then we might say that the nucleolus is “the cellular heartbeat.”

Before we delve into the functional structures that comprise the tasks of the nucleolus, let’s briefly recap the chiral theory of living awareness. As we’ve shown throughout previous chapters, unlike material objects, the cell is a living coordinate that establishes chiral (left-right) orientation and forward direction from an internal vantage point within the looping hierarchy of tasks.  A task is a relatively permanent function with a relatively temporary structure.  As functional structures through which matter passes, we’ve shown how the tasks and sub-tasks of both memory and procedure encompass chirality and forward direction at every turn.  Looking more broadly at just the tasks of memory, we see a clear mapping of the physical dimensions of matter to the chiral dimensions of life.  From this holistic perspective, nucleotides are the building blocks of one-dimensional left-right memory tasks expressing the back-and-forth functionality of the first chiral dimension.  This horizontal back-and-forth is exhibited in the left-right chirality of the molecule itself, in which the shape of the left half (the phosphate group) is different than that of the right half (the base).  From the position of the central plane of the sugar, left-phosphate and right-base map to a horizontal line.

Embedded within the nucleotide is a directional “pointer” established by its C5’-phosphate component that we’ve referred to as “up.” (As a point of reference, recall that in the left enantiomer, the C5’ atom with its attached phosphate group points downward.) This “up-facing” pointer in the building block of memory establishes an overall upward direction for the backbone of the molecule that stands in contrast to the linear end-to-end linkage we saw in the joining of successive amino acids.  This difference in the underlying shape of the building block is reflected in the shape of DNA.  Here, we have a higher-order task that expresses both 2D planar and 3D helical dimensions.  The 2D plane is reflected in the pentose sugar and the helix is reflected in the rotation of the sugar-phosphate backbone around the virtual central axis of the molecule.  Together, these two components of the nucleotide add the open-close functionality of the second chiral dimension to the back-and-forth of the first dimension.  The open-close functionality is expressed in both major and minor grooves.

Next, we saw how DNA combines with proteins in the nucleus to form a three-dimensional topological task that adds the in-out functionality of the third chiral dimension to back-forth and open-close.  In the nucleus, we see “in-out” expressed in the irregular surface topology of chromatin that establishes the means for locating the specific DNA segment of interest to the cell in any given “now” moment of living time. Now, in our examination of the nucleolus, we’ll see how the fourth chiral dimension of living time is added to these other chiral dimensions as it functions to coordinate the timing and pace of each step in the looping hierarchy of tasks throughout the cell. ²  As we enter this discussion, keep in mind that the hierarchy of tasks is a two-way flow of information about the present state of matter.  One is a forward flow into the next “now” moment; the other is a simultaneous backward flow into the feedback loop of memory.  By regulating and linking the temporal succession of both procedure-related tasks and memory-related tasks throughout the cell, the nucleolus controls this two-way flow and establishes the pace of living time.

To understand the importance of this highest-level task of the cell, we have to again look at it from the perspective of life.  Material objects in the world lack the dynamic “probing” interactions that awareness applies to matter – interactions that constitute what we’ve referred to as the “in-out” aspect of life.  Instead, they’re nothing more than intersecting surfaces onto which living awareness projects qualities that are uniquely associated with the living organism itself. An example of this type of projection is reflected in the way a human observer might describe a box as having a left and right side, a top and a bottom, as well as an “inside” and an “outside.  These characteristics reflect a unique life-centric perspective that is projected onto an inanimate object.  From the point of view of life, all three-dimensional objects have a surface that awareness integrates and remembers as “around.”  The inside / other side of such objects is a projection that living awareness makes by way of the third axis discussed in Chapter 5.

In its interactions with the world, the organism perceives a series of two-dimensional shapes gleaned from approximate left-right vantage points that spiral with motion along the third axis of awareness. It uses the information gained to project the size and shape of three-dimensional objects.  These perceived shapes are then matched to life-critical shapes stored in memory.  Those that match are “grabbed;” those that don’t are passed by without interaction.  If awareness projects and shape-matches correctly, the cell continues to live; if it does not, it dies.

The matter parts (or shapes) acquired by way of shape-matching are acted on in a successive step-by-step repeating fashion, which we’ve referred to as a functional looping hierarchy of tasks.  This hierarchy flows in two directions – forward into next “now” and backward into memory. The foundation of this task-based hierarchy “begins” with pure succession, stringing left- and right-handed building blocks one after the other. These processes ultimately establish and maintain all functional structures (tasks) of the cell that we as human observers take to constitute the material body of the organism.

The looping nature of these two-way processes reflects the fundamental curvature that is inevitably embedded in any homochiral task. Curving continuation – whether left in protein assembly or right in memory – is more than simple succession. Curvature encodes a rate of change – an angular degree that both establishes and describes the repetitional frequency of the cycle under consideration. Stated differently, rate links any sequence of curving chiral continuation to a time-based cycle. Rate is the cyclic spacing of otherwise smooth temporal succession that underlies the looping tasks performed by the cell.  As such, rate establishes the cycle-driven pace of living time.

The timing associated with any given task within the looping hierarchy applies to the entire cycle that comprises the task.  Each sub-cycle constitutes an underlying “unit” onto which awareness applies a functional rate or ”tempo of repetition.”  To understand this, consider that at its most fundamental level, awareness is change in the continual present.  Continuance without change is meaningless stasis; change without continuance (if that is even conceivable) is meaningless chaos.  Together – continuance, change, continuance, change – is timed succession with the structural integrity necessary to establish a rhythm. The interconnectivity of all tasks within the hierarchy ensures that the timing established for one task impacts that of all other tasks to the effect that every task in the cell occurs at the right time and in the right order.  Thinking of this in a purely temporal sense, we might say that every sub-task is a “fragment” of living time that combines with that of all other tasks in the looping hierarchy to constitute the integrated “now” moment of living time.  Stated differently, the organism-wide “Task of tasks” is also a “’Now’ of ‘nows’.”  The central task of the nucleolus is to regulate the frequency at which the myriad looping cycles are repeated, and to coordinate them in a way that helps ensure the organism continues into the next “now” moment.  It does this by way of ribosome production.

In the last chapter, we discussed pure succession that takes place at the beginning of amino acid assembly.  The set of tasks that translates and releases the sequence of amino acids that form the growing chain is what is objectively referred to as the ribosome. The nucleolus is responsible for transcribing the precursors of ribosomes and assembling them; it also controls when and where they’re used. This requires awareness of conditions throughout the cell at each moment of living time. The central position of the nucleolus within the structural and functional organization of the nucleus allows it to modulate all time-linked cellular processes from metabolism, cell proliferation and gene expression to the maintenance of epigenetic states and the pace of aging. ³

Although we’ll discuss ribosomes more fully in the next chapter, a brief introduction here is necessary to explain how the nucleolus functions as the master clock that regulates and controls the overall timing of tasks within the cell. To begin, ribosomes are assemblies of specialized proteins and ribosomal RNA (rRNA) that act as a unit to translate messenger RNA (mRNA) into the strings of amino acids that are the starting point of all protein production. The genes transcribed in the nucleolus are “ribosomal DNA genes” (rDNA) that code for ribosomal RNA. The genes that code for the other two principle forms of RNA – messenger RNA (mRNA) and transfer RNA (tRNA), which carries amino acids to the ribosome during translation – are transcribed in the active compartment of the nucleus, not in the nucleolus. Ribosomal RNA makes up 90% of the “nuclear transcriptome” (i.e., the precursors and initial products of DNA transcription). There are millions of ribosomes in the cell that are continually replaced after each protein is assembled. ⁴  To meet the need for transcription of such large numbers of rRNA molecules, cells contain multiple copies of the rDNA genes. The human genome contains about 200 copies of the genes that encode ribosomal RNA in the nucleolus and approximately 2,000 copies of a gene that encodes one ribosomal RNA molecule in the nucleus proper.

Ribosomal RNA is transcribed from repeating sequences of three genes arrayed in specific regions within the “short” end of chromosomes, as shown in the inset of Figure 1A. These “nucleolar organizing regions” appear in some (but not all) chromosomes, and are thought to lie in relative proximity to one another within the nucleolus after the self-similar folding of chromosomes has taken place along the nuclear scaffold. ⁵  In keeping with its central role as timekeeper of the cell, the nucleolus mediates inter-chromosomal interactions.  Having many of these “organizing regions” anchored together within the nucleolus could explain both the relative proximity of various chromosomes as well as the patterns of genomic co-localization needed to form functional TADs. ⁶

In considering the action of these nucleolar organizing regions, keep in mind that although not all chromosomes have such areas, those that don’t lie in close proximity to the ones that do. This spatial proximity (reflected in the stylized drawing of Figure 1B) allows the nucleolus to act as a common central hub.  Access to this hub may form the basis for the previously discussed spatial segregation of active euchromatin at the center of the nucleus from the more densely packed heterochromatin near the nuclear periphery.

Looking at Figure 1C, the fibrillar component of the nucleolus in yellow is the area in which ribosomal DNA is initially transcribed. Unlike genes transcribed in the rest of the nucleus, rDNA-coding genes are only transcribed in these specific areas. The surrounding border regions in purple are where the transcribed rRNA molecules are assembled.  The granular component in blue is the region from which the assembled molecules are distributed to areas beyond the nucleolus.  In thinking about this unique process for producing rRNA, we can imagine the associated rDNA being pulled by nucleosomes through a fibrillar component of the nucleolus where transcription takes place or having the processes that define such a component move through an area of rDNA located on its associated chromosome. Either way, the distribution and sequencing of the three-gene repeats in the underlying rDNA will impact the speed of transcription into the precursors of all ribosomes needed by the cell. 

As a critical functional task of amino acid assembly and protein synthesis, ribosomes are implicated in the gamut of intracellular activity – from genomic translation to organism-level recognition and environmental interaction. This cell-wide role makes them the natural foci for coordinating the timing of all tasks that comprise the living cell. The combination of their conveyor-belt-like function in amino acid assembly plus the cyclic nature of their production in the nucleolus (described below) allows ribosomes to establish a unified tempo of cellular life.  This tempo (or “rate of repeat”) refers to a global timing that determines when succession (expressed in the one-by-one stringing of amino acids and the nucleotide-by-nucleotide transcription of DNA) can take place. While this global rate establishes the broad temporal parameters within which local processes can occur, it is the local factors themselves which determine how quickly the sub-tasks of succession can be completed.  Such factors include the nature of the underlying DNA code, the length of the amino acid chain and the action of nucleosomes involved in transcription.  For example, with respect to nucleosomes, the eight-unit clock determines the spacing of alternating periods of unwinding / pull and release / delay during transcription.  This spacing directly impacts how fast mRNA molecules can present the products of transcription for translation by the ribosome in the cytoplasm of the cell.  As this example shows, the commonality of succession across all cellular processes and the mutual dependence of those processes on transcription, translation and protein assembly means that the rates that apply to small embedded cycles in the process of ribosome production affect the global rate at which all other intracellular processes can take place. 

Looking more specifically at ribosome production, the general pathway consists of three main stages: rRNA synthesis, processing, and assembly. To accomplish these tasks, the nucleolus must accumulate the information / sub-tasks needed for each step of the process, generate the precursor molecules by way of rDNA transcription, and integrate and package those “transcripts” into completed units for export into the cytoplasm of the cell.  Clearly, these steps must be completed in a specific order. Transcription proteins must be available before rDNA transcription can begin.  Assembly must wait until the RNA precursor molecules have been transcribed and ribosomal proteins have been “recruited” for packaging.  The export of finished ribosome units can’t take place until the proteins and precursors have been assembled and packaged.  As these information-dependent processes take place, they cause the nucleolus to “pulse,” variously slowing down and speeding up, contracting and expanding, as it first accumulates and then acts on the small structures required for each step of the process.  These pulses form a rhythm in living time, linked to the repeating cycles that encompass rDNA transcription, precursor genesis, sub-unit assembly and export. ⁷

Let’s examine each step of the ribosome-production process in a bit more detail. Ribosome genesis begins with the transcription of the three rDNA genes as one unit. ⁸  During the assembly and processing stage, the resulting precursor RNA is packaged together with specific proteins to make two sub-units – one large and one small – that are further combined to form the two halves of a finished ribosome. This process is represented in the stylized flow shown in Figure 2. Here, green arrows represent the flow of rRNA into the ribosomes and red arrows show the flow of proteins into and out of ribosomes as well as into other tasks of the cell, including the nuclear membrane, cytoplasm and cell membrane.   At the top of this diagram, the three-gene rDNA array is shown, representing the transcription site that codes for the RNA precursor molecule.  The purple circle and arrows indicate the path of transcription along the rDNA.  Ribosomal RNA precursors are indicated by the small green and red pentagons; ribosomal proteins are indicated by green and red squiggles.

Once assembly and packaging is complete, the ribosome is dispatched through the nuclear membrane and into the cytoplasm of the cell. There, it undergoes various “quality control” tests before it’s released to translate all the many proteins produced by the cell. This is where the two sub-units of the molecule come into play.  The large one (shaded light blue in Figure 2) processes tRNA, which transports amino acids to the site of translation; the small one (shaded darker blue) processes the mRNA codon. Once a ribosome has been used in translation, it’s disassembled and its parts are recycled. As an indication of the complexity involved in the total process, consider that each finished ribosome is comprised of nearly 7,000 nucleotides, and each rDNA transcription unit is about 13,000 bases long. ⁹ 

As we might predict, the rate of ribosome production and protein translation are tightly coordinated. There’s an optimal concentration of ribosomes at which protein synthesis is maximal, and beyond which translational efficiency is impaired.¹⁰   To achieve the right balance, ribosome transcription, assembly, export and testing cycles feedback into a larger meta-cycle involving three RNA-related enzymes (referred to as RNA polymerases or RNAPs). One of these polymerases (RNAP II) transcribes mRNA, and another (RNAP III) transcribes tRNA. Only RNAP I transcribes rRNA within the nucleolus. The synchronized activity of these three enzymes constitute an over-arching self-regulating process within which smaller cycles loop to ultimately control and be controlled by the repeating cycles of translation and transcription. The flow of mRNA and tRNA from DNA into and out of ribosomes is indicated by the blue arrows in Figure 2.

Since ribosomes are required for mRNA and tRNA to function, the timing of their production in the nucleolus impacts every metabolic pathway within the cell.  The relative speeding and slowing of ribosome production in response to conditions within the nucleolus establishes the global rate of repeat that applies to succession, as described above.  This rate applies to the organism as a whole, limiting when the sequential hand-off of embedded sub-tasks in the looping hierarchy can take place. This, in conjunction with the tri-part action of RNAPs, serves to balance the cell-wide activities of transcription with the cell-wide activities of translation.  Let’s look more closely at how this homeostatic balance is achieved. 

Essentially, cellular homeostasis is a dynamic balancing of the flow of information into and out of the nucleolus.  By “information,” we mean RNA precursors, proteins, and the products of related sub-tasks that pass into and out of the nucleolus, causing it to undergo alternating cycles of expansion and contraction.  Since the nucleolus has no membrane, it is this size-linked functionality rather than any specific structure that largely defines the nucleolus relative to the rest of the nucleus. We see the emphasis on function in Figure 2, which serves as more of a flow chart than a static depiction of the properties of a solid structure. 

The relationship between information flow and the physical size of the nucleolus is made possible precisely because of the looping hierarchy of tasks we’ve described throughout this book.  Any given part of a closed loop will expand with reduced out-flow and contract or shrink with increased out-flow.  This is the same size-to-rate relationship we see in the appearance and disappearance of bottlenecks in all dynamic processes from traffic flow to localized flooding of rivers and streams.

Awareness uses the in-out flow of information through the nucleolus to control the cell-wide cycles of succession that establish the pace of living time.  It does so by linking information flow through the nucleolus to the timing and speed of ribosome production. When nutrients in the nucleolus are restricted or in short supply, more information (proteins, rRNA precursors, etc.) flows out than in.  The nucleolus responds by shrinking in size and becoming more efficient in its use of available supplies, making more ribosomes in less time. On the other hand, if more information flows in than out, the nucleolus expands, becoming larger and slower, producing fewer ribosomes until ultimately, production halts and cell division occurs. ¹¹   By regulating ribosome availability in this way, the nucleolus establishes periods of mRNA translation and thus exerts control over procedure. The resulting regulation of access to transcription proteins simultaneously allows the nucleolus to control the activities of nucleosomes and transcription, and thus to exert control over memory.  Together, these complementary processes strike a balance between growth / self-maintenance / self-replacement and cell division. ¹²   In Figure 2, this balance is reflected by green arrows showing the flow of rRNA into ribosomes and out into the cell matrix where they’ll perform the translation tasks of procedure, and by red arrows showing the flow of proteins and related information from ribosomes back into the nucleus and nucleolus where they’ll perform the transcription activities of memory and cellular timing.  Black arrows encircling the perimeter of the cell indicate the flow of information by way of proteins back into the nucleolus from all parts of the cell cytoplasm and every point along the cell membrane. 

Notice that all the cycles embedded in the hierarchy of tasks are circular and self-referential.  The green, red and blue arrows of out-flow are counterbalanced by red and black arrows of in-flow.  Ribosomes make the proteins that that are needed to make ribosomes; DNA codes for proteins that update and repair DNA; interphase precedes cell division and cell division precedes interphase… From the logical standpoint of matter time, such circularity is a causative contradiction; that which is caused cannot cause itself. However, from the standpoint of life, no contradiction exists.  In each “now” moment, the present state of matter is projected forward into the next “now” moment.  As “now” transitions into next “now, an updated record of the current status is simultaneously transferred backward for storage in memory.  Throughout it all, living time remains constant in the persistent “now.” 

The homeostatic process we’ve just described addresses all the activities that take place within the cell.  But these activities don’t take place in a vacuum.  The cell lives in and interacts with the world.  How does life achieve homeostasis within the ever-changing dynamic environment in which it lives? The answer to this question brings us to the largest cycle in the looping hierarchy of tasks – one that links activity within the nucleolus to the world in which the cell lives – a world that exists, evolves and changes in the context of time.  As we’ve said elsewhere, time is pure succession.  In the external world, light and dark establish the repeating cycles of succession.  In life, living time establishes the repeating cycles of succession.  Life uses the three languages of transitional dimensionality – directional lines, shapes and symbols – to temporarily link functional structures generated by a looping hierarchy of tasks to the circadian rhythm.  In the process, it generates a shape-based means of interacting with the world from a temporospatial coordinate of living time.  By establishing its own timing cycle linked to that of the external world, the tasks of the cell exist in the context of matter time even as they also exist in contrast to matter time.  At every level of the looping hierarchy, the cycles of tasks and sub-tasks extend outward and beyond the present moment, even as they ever remain fully within it.  By seeding the next “now” with elements of the current “now,” life recreates the immediate past in the new “now” moment and propels itself ever onward in matter time, maintaining a self-same rhythm predicated on a constant left-right-forward orientation in the eternal “now” moment.

The nucleolus plays a critical role in establishing this dynamic relationship between the living cell and the environment in which it operates.  It does so by linking the cell-wide repeating cycles of transcription and translation activities to the circadian rhythm. Taking the cells of nocturnal animals as an example, the nucleolus reaches its maximum size during the day – expanding to reflect a net inward flow of information into the nucleolus.  This period of accumulation and expansion is then followed by a period of net information out-flow during the overnight hours, reflected in a rapid shrinking of the nucleolus as ribosomes are exported into the rest of the nucleus and out into the cell cytoplasm or beyond.  As daylight returns, the cycle repeats, with the nucleolus again expanding in a net in-flow of information.  This direct response to sunlight gives the nucleolus an intrinsic link to the larger external cycle of light and darkness, allowing the cell to coordinate its looping tasks with the diverse environmental conditions associated with the 24-hour circadian cycle. ¹³  As a result, the nucleolus not only controls the repeat rate of looping sub-tasks, but also acquires information that allows the cell to anticipate and therefore predict certain environmental changes that, in turn, increase its chances of survival into the next “now” moment.

We opened this discussion of the nucleolus with a brief recap of the functional structures of memory (the right nucleotide, the DNA double helix and the nucleus) as they relate to the chiral dimensions of life. Before closing this chapter, let’s briefly re-cap how these three chiral dimensions – 1D back-forth, 2D open-close and 3D in-out – are embedded in the time-related tasks of the nucleolus.  As we’ve said, tasks are functional structures that operate within the flow of living time.  The present status of the cell at any “now” moment is information received from the immediate past ”now” that is acted on and passed forward into the next “now” moment.  In this way, the forward flow of living time links the instant-to-instant physical structure of tasks to the instant-to-instant motions of shape-fitting. As a result, the shape-related aspects of transcription are synchronized with the shape-related aspects of translation on both a global and local level.  Globally, transcriptional proteins shape-fit chromatin to locate the specific surface of DNA-base pairs to be transcribed; that surface is then opened and transcribed locally at a pace established by the nucleosome clock.  Globally, the surface of millions of available ribosomes scattered throughout the cell cytoplasm impact the time-linked efficiency with which individual ribosomes locally translate mRNA codons to amino acids that fold into proteins.

In the course of these interrelated global-local processes, the first chiral dimension (backward-forward) is expressed in the feed-forward / feed-back “turning around” of past matter to forward-facing information. The forward flow of ribosome genesis, assembly and export controls the production of proteins that in turn, capture and carry information on the status of the intra- and extra-cellular environment back to the nucleolus in a feedback loop.  In both protein assembly and DNA structure, growth is added from behind, not by new accumulation at the front. In the case of amino acids, the chain grows from the rear as each new amino acid adds length to the already-assembled chain.  In the case of memory, the store of past information grows as each new “now” moment adds its content to the growing cache. 

The nucleolus expresses the second chiral dimension (open-close) in alternating periods of succession and delay timed to the organism-wide rate of cyclic repeat.  In the nucleolus, succession takes place during periods of net information in-flow when the genome is open for the transcription of rDNA nucleotides.  These are followed by periods of ribosome assembly and export, when the genome is closed until succession begins anew in the next timed period of cyclic repeat.  These global pulses of succession and delay in the nucleolus are reflected locally in the topologically chiral pull-and-release motions of nucleosomes operating on the DNA strand in the rest of the nucleus.

The third chiral dimension (in-out) is expressed as a change in the size of the nucleolus over the course of the longest looping cycle of tasks performed by the organism. These are the tasks linked to the 24-hour circadian cycle, described above.  During this cycle, the size of the nucleolus alternately expands outward in a period of gene transcription and precursor genesis reflecting a net influx of information.  When maximum size is reached, it then begins a period of contraction inward as ribosomes are packaged and exported, reflecting a net out-flow of information into the nucleus, cytoplasm and beyond. 

¹   While DNA contains the instructions for re-creating the status of life in each new “now” moment, it’s the job of the nucleolus to ensure everything works well and on time.

²   The nucleolus is scientifically described as a “multiphase condensate.”  It is a liquid-like state.  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721831/

³   https://pubmed.ncbi.nlm.nih.gov/10585627/

⁴   https://bscb.org/learning-resources/softcell-e-learning/ribosome/

⁵   https://en.wikipedia.org/wiki/Nucleolus_organizer_region

⁶   The structural organization of the nucleolus is still a subject of scientific investigation.  It’s possible that each of the organizing regions we’ve described might have its own fibrillar component. Not all chromosomes contain such regions, and of those that do, only some are transcriptionally active.  Moreover, chromosomes that do not contain these areas can and do contain transcribable genes. 

⁷   https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555582/

⁸   https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8559705/

⁹   https://en.wikipedia.org/wiki/ribosome

¹⁰ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4233708/

¹¹ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7123373/ 

¹² https://www.cell.com/trends/cell-biology/fulltext/S0962-8924(18)30063-1 

¹³  Animals that live in total darkness retain thermo-regulatory rhythms.  See https://www.pnas.org/doi/10.1073/pnas.181484498