Methods to characterize the compositional and functional histories of cellular proteins and associated macromolecular complexes in space and time

Abstract

Disclosed is a method for characterizing compositional and functional histories of cellular proteins and their associated molecular complexes in space and time. The method may include forming fusion proteins by fusing a universal genetic tag to each of a plurality of endogenous proteins. The fusion proteins may be conditionally labelled with a first ligand. The first ligand may be a ligand with a developed immunopurification capability and a known imaging capability. The method may include expressing the fusion proteins and performing at least one analysis. The analyses may include (i) tracking the plurality of endogenous proteins and any associated macromolecular complexes; (ii) determining composition and function of the plurality of endogenous proteins and / or any associated macromolecular complexes; and / or (iii) removing a ligand and performing a pulse-chase analysis.

Description

[0001] METHODS TO CHARACTERIZE THE COMPOSITIONAL AND FUNCTIONAL HISTORIES OF CELLULAR PROTEINS AND ASSOCIATED MACROMOLECULAR COMPLEXES IN SPACE AND TIME

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] The present application claims priority to U.S. Provisional Patent Application No. 63 / 543,329, filed October 10, 2023, the contents of which are incorporated by reference herein in its entirety.

[0004] TECHNICAL FIELD

[0005] The present disclosure is drawn to techniques for characterizing the compositional and functional histories of cellular proteins and associated macromolecular complexes in space and time.

[0006] BACKGROUND

[0007] Cellular proteins and protein complexes are in a dynamic state of biogenesis and turnover. The balance between these two processes is critical for cellular homeostasis, with imbalances being hallmarks of diseases of aging and cancer. Furthermore, they can be subjected to post-translational processing and modifications that alters their functions over time. However, little is known about the dynamics of proteins and associated macromolecular complexes in vivo, as there are currently limited methods that can distinguish old proteins from new proteins. There are currently no methods that allow purifying old and new proteins and associated complexes in native conditions and simultaneously resolve their spatiotemporal organization.

[0008] BRIEF SUMMARY

[0009] In various aspects, a method for characterizing compositional and functional histories of cellular proteins and their associated molecular complexes in space and time may be provided. The method may include forming fusion proteins by fusing a universal genetic tag to each of a plurality of endogenous proteins. The method may include expressing the fusion proteins and performing at least one analysis. The analyses may include (i) tracking the plurality of endogenous proteins and any associated macromolecular complexes using a ligand that binds to the fusion proteins; (ii) determining composition and function of the plurality of endogenous proteins and / or any associated macromolecular complexes; and / or (iii) removing a ligand from a cell feeding media, where the cell feeding media is used to perform a chase in a pulse-chase analysis, as new proteins won’t be able to bind the (now removed) ligand.

[0010] The analyses may vary depending on whether you wish to track pre-existing complexes, nascent complexes, or both. To track pre-existing complexes: 1) cells are fed with media containing the first ligand (the “cell feeding media”), 2) the disclosed fusion proteins bind the first ligand covalently, and 3) washout of the first ligand ensures no new proteins bind the ligand and one can track then the fate of pre-existing proteins over time. To track nascent complexes: 1) cells are fed with a blocking ligand (such as a non-fluorescent blocking ligand, or a blocking ligand that fluoresces at a different wavelength than the first ligand) that saturates all pre-existing complexes, 2) the blocking ligand is then washed off the media and the first ligand is then fed to the cells, such that only nascent protein that was not blocked with the blocking ligand can be bound to the first ligand and analyzed.

[0011] The method may include recording a compositional and functional history of the plurality of endogenous proteins in space and time. A CRISPR-based technique may be used to fuse genetic tags to the plurality of endogenous protein. Tracking may be accomplished via live-cell confocal microscopy. The composition and function may be determined by immunopurification followed by quantitative mass spectrometry and / or genomic analysis.

[0012] The method may include providing at least one of the fusion proteins to each of a plurality of containers, exposing each container to one or more chemical or biological agents, and then identifying a chemical or biological agent that specifically targets an old or a young protein by performing the at least one analysis.

[0013] In various aspects, a kit may be provided. The kit may include a universal genetic tag; and a first ligand, the first ligand (such as a halo tag ligand) being a ligand with a developed immunopurification capability and a known imaging capability.

[0014] BRIEF DESCRIPTION OF FIGURES

[0015] Figure 1 is a flowchart of a method.

[0016] Figures 2A is a schematic illustration of a fusion protein complex with a first ligand.

[0017] Figure 2B is a schematic illustration of a first ligand.

[0018] Figure 2C is a schematic illustration of a fusion protein complex with a blocking ligand.

[0019] Figure 3 is a schematic illustration of a nucleotide sequence expressing a protein construct.

[0020] Figure 4 is a flowchart of a method.

[0021] Figure 5 is a schematic illustration of a system. Figure 6 is a schematic illustration of the haloalkane dehalogenase tag / haloalkane ligand irreversible binding mechanism.

[0022] Figure 7 is a schematic illustration of pulse-chase experiments using a haloalkane dehalogenase tag.

[0023] Figure 8 provides a diagram for recording spatiotemporal dynamics of nascent and preexisting spliceosomes, as well as sample images captured using the technique.

[0024] Figure 9 is a schematic illustration of a summary of some experimental tools and strategies that may be used as part of the disclosed technique.

[0025] Figure 10 is a diagram showing RNA Tapestation and Western Blot results for various proteins, using the disclosed techniques versus a control.

[0026] Figure 11 is a schematic illustration of techniques for visualizing how (or whether) a ‘steady-state’ interactome may be built over time, demonstrating that mass spectrometry analysis allows determination of the steady state interactome of ligand-mediated purified complexes

[0027] Figure 12 is a diagram illustrating a haloalkane dehalogenase tag pulse-chase paradigm moving toward steady-state.

[0028] Figure 13 is a diagram that illustrates recording cytoplasmic 40S maturation and function, as well as sample images captured during the process.

[0029] Figure 14 is a plot demonstrating the relative fraction of the steady state 40S population is constituted by the nascent population being probed as quantified by microscopy

[0030] Figure 15 is a plot showing protein expression fold changes captured using a disclosed construct, versus with a simple haloalkane dehalogenase tag control, from ligand-mediated immunopurification data.

[0031] Figure 16 is a bar chart showing the percentage of various subunits captured in cytoplasmic 40S immunoprecipitation (IP).

[0032] Figure 17 is a plot showing gene ontology for cytoplasmic 40S IP.

[0033] Figure 18 is a plot of nascent small subunit (SSU) ribosomal proteins enriched over time, as a percentage of steady state.

[0034] Figure 19 is a plot of nascent large subunit (LSU) ribosomal proteins enriched over time, as a percentage of steady state.

[0035] Figure 20 is a plot of nascent small subunit (SSU) assembly factors enriched over time, as a percentage of steady state.

[0036] Figures 21A and 21B are plots of eukaryotic translation initiation factor 3 subunits enriched over time, as a percentage of steady state. Figure 22 is a plot of eukaryotic translation initiation factor 4 subunits enriched over time, as a percentage of steady state.

[0037] Figure 23 is a plot of plots of eukaryotic elongation factor subunits enriched over time, as a percentage of steady state.

[0038] Figure 24 is a diagram that illustrates recording nucleolar pre-60S assembly, as well as sample images captured during the process.

[0039] Figure 25 is a plot showing, for an average of around 700 nucleoli, the large ribosomal subunit protein (RPL) core enrichment and rim enrichment over time.

[0040] Figure 26 is a plot showing pre-existing pre-60S ribosomal proteins over time, as a percentage of steady state.

[0041] Figure 27 is a plot showing pre-existing pre-60S assembly factors over time, as a percentage of steady state.

[0042] Figure 28 is a plot of pre-existing nucleolar proteins over time, as a percentage of steady state.

[0043] Figure 29 is a plot showing the early depletion of certain nucleolar proteins over time, as a percentage of steady state.

[0044] Figure 30 is a diagram and set of images that describes the ability of performing a pulsechase strategy to distinguish pre-existing and nascent snoRNP complexes in HEK293 cells via aNoppl40-HaloTag fusion.

[0045] DETAILED DESCRIPTION

[0046] There are currently no methods that 1) distinguish pre-existing complexes from nascent complexes, 2) allows purifying these complexes in native conditions for compositional analysis, and / or 3) allows visualizing those complexes of select ages by microscopy all together via one comprehensive method.

[0047] Disclosed herein are methods to characterize the compositional and functional histories of cellular proteins and associated macromolecular complexes in space and time. The disclosed method tackles, e.g., the problem of distinguishing the spatiotemporal organization and composition of old proteins from new proteins, by utilizing a universal genetic tag that can be conditionally labeled with a ligand. The protein can then be specifically immunopunfied using the tag, as well as imaged to monitor its subcellular localization. This allows taking intracellular snapshots by confocal microscopy, as well as immunopurification as a function of age. These purified proteins and complexes can be subjected to quantitative mass spectrometry and genomics analyses for assessing their composition and function. Together with live-cell imaging, this method provides an in vivo spatial, compositional, and functional history of the protein or complex of interest.

[0048] The disclosed method can be used for the recording of histories of various macromolecular complexes in physiological and diseased conditions. These will provide a glance into their spatial, compositional and functional evolution as a function of age and the impact of altered cellular conditions on these histories.

[0049] Characterizing the composition and function of intracellular proteins in space and time can inform about fundamental differences between healthy and diseased cellular states. To date, this remains an underappreciated dimension of protein function for which tools to study are lacking. Future applications may screen drugs for their effects on these dynamics on specific cellular proteins within distinct cellular contexts to target disease. For example, the disclosed technology could be deployed in a high throughput screening platform for identifying compounds that specifically target old or young proteins, with potential implications for aging or age-related protein diseases. As a simpler example of its use, one could imagine a kit in which a set of halo tag ligands are utilized for initial labeling, blocking, and subsequent pulsing etc.

[0050] This is understood to be the first process that can record the compositional and functional histories of cellular proteins in space and time. The use of a universal genetic tag allows for a high degree of modularity that would make this process accessible to most of the cellular proteome. This process has the potential to opening the study of an understudied dimension of cellular protein function - molecular age. Furthermore, this strategy could also be used to characterize the assembly of nascent macromolecular machines, such as ribosomes or spliceosomes.

[0051] With the disclosed process, one fuses genetic tags to endogenous proteins (e.g., using CRISPR-based strategies) and conditionally labels them with a ligand for which has been developed immunopurification capability, in addition to its known imaging capability. This allows tracking of endogenous proteins and their associated macromolecular complexes by live- cell confocal microscopy as well determination of composition and function by immunopurification followed by quantitative mass spectrometry or genomic analyses. Because the labeling of the genetic tag is conditional on the presence of ligand, removal of the ligand from the cells / media allows for pulse-chase experiments that facilitates tracking the spatial, compositional, and functional history of the protein of interest via aforementioned methods. Applying this process to proteins of low abundance requires large amounts of native lysate which may be costly to prepare depending on the cell type or tissue from which material is being extracted.

[0052] Referring to FIGS. 1-2C, the method (100) may include providing (110) a desired protein construct (220) or a sequence for expressing a desired protein construct (220). As part of the disclosed modular process fusion proteins (220) are formed using proteins of interest, such as endogenous proteins (210) for a given organism, and genetically tagging them with a universal genetic tag (220). The universal genetic tag is a tag (such as a haloalkane dehalogenase tag) that covalently binds to a first ligand (224). Referring to FIG. 2B, the first ligand (224) is a ligand that generally includes two parts - a binding portion (226) that specifically binds to the universal genetic tag (for a haloalkane dehalogenase tag, the binding portion would include a haloalkane ligand that binds to the tag), and a fluorescent portion (228) that is ty pically a dye or other component that can be used to track the ligand. The disclosed method takes advantage of that universal genetic tag - specific ligand binding to form complexes (200) that are useful. In FIG. 2A, the first ligand (224) would include a dye or marker (such as a tetramethylrhodamine (TMR) dye) that allows the components that the first ligand covalently binds with to be tracked. In FIG. 2C, the first ligand is replaced with a blocking ligand (230). The blocking ligand may be, e.g., a ligand that only has the, e.g., binding portion (228) of the first ligand, that binds to the universal genetic tag. The blocking ligand may include a fluorescent portion (228), but emit a different wavelength from that used by the first ligand.

[0053] The fusion proteins may include a cleavage tag (221) disposed between the protein of interest (e.g., endogenous protein (210) and the universal genetic tag (222). The cleavage tag (221) may be any appropriate known cleavage tag. Such cleavage tags include, e.g., selfcleaving tags, Human Rhinovirus 3C Protease (3C / PreScission), Enterokinase (EKT), Factor Xa (FXa), Tobacco Etch Virus Protease (TEV), or Thrombin (Thr)cleavage tags

[0054] Fusing may include using CRISPR-based strategies.

[0055] Referring to FIG. 6, an example of this may considered. In FIG. 6, a protein of interest (POI) (614) is fused to a haloalkane dehalogenase tag (610), where the haloalkane dehalogenase tag includes a cleavage site (such as a TEV cleavage site). Haloalkane dehalogenase tags bind haloalkane ligands irreversibly, the haloalkane ligand forming a covalent bond (630) with the haloalkane dehalogenase tag when the halogen (here, a chlorine ion) is removed. Referring to FIGS. 2A-3, a nucleic acid sequence (300), such as an isolated polynucleotide sequence, may be provided, where a polynucleotide sequence (320) configured to express the fusion protein (220) may be placed under the control of a promoter (310), such as an endogenous promoter. Any endogenous promoter may be utilized.

[0056] The universal genetic tag may be any appropriate universal genetic tag. As is known in the art, a universal genetic tag is a molecular marker that can be used to label and track proteins across different systems, organisms, or experimental conditions. The goal of such tags is to provide a consistent and versatile method for monitoring protein expression, localization, or interactions regardless of the specific context in which they are used. Such tags are considered "universal" because they can be applied to various experimental systems and provide reliable and reproducible results. They facilitate protein manipulation and analysis across different research contexts.

[0057] Such universal genetic tags may be, e.g., self-labelling protein tags. Self-labelling protein tags with imaging capability are designed to bind to small molecules or ligands that can be visualized using imaging techniques such as fluorescence microscopy. These tags allow researchers to study protein localization, dynamics, and interactions in live cells or tissues without the need for secondary antibodies. Non-limiting examples of universal genetic tags include, e.g.:

[0058] HALOTAG® protein tag: a protein tag derived from the Haloalkane Dehalogenase enzyme. It covalently binds to a synthetic ligand or substrate that is conjugated to a fluorophore or other imaging probe. Various fluorophore-conjugated HALOTAG® ligands are available, enabling live-cell imaging and tracking of proteins with high specificity.

[0059] SNAP -tag: a protein tag based on the O6-alkylguanine DNA alkyltransferase enzyme. It binds specifically to a synthetic ligand containing a fluorophore or other imaging moiety. SNAP-tag ligands are available with a variety of fluorophores, allowing for multi-color imaging and live-cell studies.

[0060] CLIP-tag: a variant of the O6-alkylguanine DNA alkyltransferase, optimized for binding to different synthetic ligands than SNAP-tag. Similar to SNAP-tag, CLIP-tag ligands are available with various fluorophores, enabling fluorescent imaging of proteins in live cells.

[0061] Lipoic Acid Ligase (LplA) Tag: a protein tag that utilizes the Lipoic Acid Ligase enzyme, which binds to lipoic acid denvatives. When tagged proteins are expressed, they can be visualized using lipoic acid derivatives conjugated to fluorescent dyes. Fluorescently labeled lipoic acid derivatives enable imaging of proteins in live cells. BioID and Proximity Labeling Tags: While not strictly self-labelling in the traditional sense, BioID tags use a biotin ligase enzyme to biotinylate nearby proteins, which can then be visualized using fluorescently labeled streptavidin. The biotinylated proteins can be detected with fluorescently labeled streptavidin, allowing for imaging of protein-protein interactions and protein localization.

[0062] The first ligand may be any appropriate ligand with a developed immunopurification capability and a known imaging capability, that can bind to the universal genetic tag. As disclosed herein, the universal genetic tags (222) and their associated binding portions are (226) well known in the art. As shown in FIG. 2B, the first ligand may also include a fluorescent portion.

[0063] The fluorescent portion may include a fluorescent dye. The fluorescent dye may be any appropriate known fluorescent dye, including fluorescein dyes, rhodamine dyes (a preferred dye being tetramethylrhodamine (TMR) dye), Cy3 and Cy5 dyes, ALEXA FLUOR® dyes (such as ALEXA FLUOR® 488 dye, 594 dye, or 647 dye), or Dylight dyes.

[0064] The fluorescent portion may include a fluorescent protein (such as green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP) (such as mCherry), etc.

[0065] A critical requirement here that for a particular fluorescent portion (such as a fluorescent dye) to be used, there must be a way to immunopurify it. Some immunopurification techniques may require high affinity antibodies. For example, tetramethylrhodamine-bound HaloTag-fusion complexes may be immunopurified by producing superparamagnetic beads covalently fused to high affinity anti-tetramethylrhodamine antibodies. These allow immobilization of complexes in these beads, performing washes and eluting off the beads either by denaturation or protease cleavage (TEV, etc). A similar approach can be used for other dyes for which high affinity antibodies are available or can be developed.

[0066] These substrates have the capability of live-cell confocal microscopy as well as immunopurification for subsequent compositional and functional characterization by quantitative mass spectrometry and genomics. Together, these allow tracking the composition and function of any protein and associated macromolecular complex in space and time.

[0067] As will be understood, in certain aspects, an expression cassette may be provided, which may comprise: an isolated polynucleotide (e.g., polynucleotide sequence (320)) that encodes the protein construct (200) as disclosed herein, and a heterologous promoter (e.g., promoter (310)), wherein the heterologous promoter is functionally linked to the isolated polynucleotide. In some embodiments, a cloning or expression vector may be provided, which may include at least one such expression cassette.

[0068] Referring to FIGS. 1-3, in some embodiments, the fusion protein (220) or nucleic acid sequence (300) may then be introduced (120) to a cell in any appropriate manner (e.g., the sequences may be introduced via transfection, using a viral vector, etc.). The desired protein fusion proteins may then be expressed (130) in the cell, for example via natural or artificial inducement of the expression of the fusion proteins.

[0069] The method may include analyzing (140) the proteins and / or recording (150) a compositional and functional history of the plurality of endogenous proteins in space and time. In some embodiments, analysis (e.g., part of the analyzing (140) step) may be done, or may utilize, the recorded history.

[0070] One or more analyses may be utilized. In some embodiments, the analysis step may include tracking (142) the plurality of endogenous proteins and any associated macromolecular complexes. Tracking may be accomplished via live-cell confocal microscopy.

[0071] In some embodiments, the analysis step may include determining (144) composition and function of the plurality of endogenous proteins and / or any associated macromolecular complexes. This may be done in any appropriate manner known to those of skill in the art. For example, this may include:

[0072] Protein Isolation and Purification, including, tissue extraction (e.g., isolating the protein from the biological source (e.g., cells, tissues)), such as via homogenization and centrifugation), and chromatography (e.g., using affinity chromatography, ion exchange chromatography, or gel filtration to purify the protein of interest);

[0073] Protein Characterization (e.g., via SDS-PAGE, Western Blotting, and / or Mass Spectrometry (MS));

[0074] Protein Sequencing (e.g., via Edman degradation, to sequentially remove and identify amino acids from the N-terminus of the protein), and MS, such as Tandem MS to sequence peptides derived from the protein);

[0075] Structural Analysis (e.g., via x-ray crystallography to determine the three-dimensional structure of a protein at atomic resolution by analyzing diffraction patterns of protein crystals, Nuclear Magnetic Resonance (NMR) Spectroscopy to provide information on the protein’s structure in solution, and / or cryo-electron microscopy (Cryo-EM) to capture high-resolution images of proteins frozen in their native state);

[0076] Functional Assays (e.g., via enzyme activity assays, binding studies, and / or gene knockout / knockdown); Bioinformatics Analysis (e.g., using sequence alignment to compare the protein sequence with known sequences to predict functions based on homology, and protein databases: Use databases like UniProt or Protein Data Bank (PDB) to gather information on protein functions and structures); and

[0077] Functional Genomics (e.g., gene expression studies to analyze how the protein’s gene is regulated under various conditions using techniques like quantitative PCR or RNA sequencing, and / or proteomics to study the protein’s interactions with other proteins, lipids, or nucleic acids within the cell).

[0078] As will be understood, the choice of methods often depends on the protein’s characteristics and the specific research questions.

[0079] The analysis step may include removing (146) a ligand from cell feeding media, and may include performing apulse-chase analysis using the plurality of endogenous proteins. Such techniques are well understood in the art.

[0080] Thus, there are two techniques that can be used, depending on what components (preexisting elements within a cell, or nascent elements of a cell) one wishes to monitor over time.

[0081] Tracking pre-existing complexes generally includes: (1) feeding cells with media containing the first ligand and allowing the fusion proteins to bind the first ligand covalently; (2) washing out the first ligand to ensure no new proteins bind the ligand; and (3) tracking the fusion proteins bound to the first ligand over time.

[0082] Tracking nascent complexes generally includes: (1) feeding cells with media containing a blocking ligand and allowing all pre-existing complexes to be saturated with the blocking ligand; (2) washing off the blocking ligand; (3) feeding the first ligand to the cells; and (4) tracking nascent proteins bound to the first ligand.

[0083] Referring to FIG. 7, a schematic of this is seen, first ligands are used to label preexisting I parental population to saturation. After one or more washouts, a second ligand can be used to label nascent populations continuously.

[0084] Referring to FIG. 8, a further example of this can be seen. Here, human bronchial / tracheal epithelial cells (HBECs) were modified to produce haloalkane dehalogenase tag - Small nuclear ribonucleoprotein-associated proteins B and B1(SNRPB). Specifically, a HALOTAG® protein tags were used to create HALOTAG® - SNRPB fusion proteins, expressed by the cells.

[0085] At time t=0 hours, cells were fed with a non-fluorescent blocking ligand that saturated all pre-existing complexes. After 30 minutes, a washout occurred (washing the non-fluorescent blocking ligand off the media), following by a 30 minute blocking period and a second washout. A ligand of interest (second ligand) was then then fed to the cells, such that only nascent protein that was not blocked with the non-fluorescent ligand was bound and analyzed / tracked over time. This can be seen in the bottom series of images over time t=0 to t=12 hours.

[0086] In some embodiments, composition and function are determined by immunopurification followed by quantitative mass spectrometry and / or genomic analysis.

[0087] Referring to FIG. 4, the method (100) may also include using the fusion proteins to identify agent that specifically targets an old or a young protein. As used herein, the term “young protein” and “old protein” refers to the relative times in which a protein is synthesized during a process. A young protein may be considered on a relative basis as, e.g., one of the top 50%, top 33%, top 25%, or top 10% most recently synthesized fusion proteins existing at a location at any point in time. Alternatively, a young protein may be considered on an absolute basis as, e.g., a labelled protein that was synthesized less than, e g., a predetermined threshold of time such as 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes, 1 minute, 30 seconds, 15 seconds, or 10 seconds in the past.

[0088] Similarly, an old protein may be considered, e.g., one of the bottom 50%, bottom 33%, bottom 25%, or bottom 10% synthesized fusion proteins of the most recently synthesized fusion proteins existing at a location at any point in time. Alternatively, an old protein may be considered on an absolute basis as, e.g., a fusion protein that was synthesized more than, e.g., a predetermined threshold of time such as 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hours, 2 hours, 3 hours, or 4 hours in the past. These techniques have been proven to work even over extended times, including over several days (e.g., over 24 hours, 48 hours, 60 hours, 72 hours, or 96 hours). Thus, these techniques allow the ability to probe features over very long molecular timescales.

[0089] The method may therefore include providing (410) at least one of the fusion proteins to each of a plurality of containers. The method may include exposing (420) each container to one or more chemical or biological agents. The method may include identifying (430) a chemical or biological agent that specifically targets an old or a young protein by performing the at least one analysis (140).

[0090] An example of a system for this may be seen in FIG. 5, where containers (510) (here, the wells in a 2x2 array of wells) may be provided. Proteins (512) may be disposed within each container (510). The proteins may be directly added to the containers, or cells may be grown within each container, where the cells have been modified to express the disclosed fusion proteins. One or more chemical or biological agents (514) may be added to the containers. As will be understood, one or more containers may be kept as a control.

[0091] A camera (520) may be disposed above the containers, and at least one lens (522) may be used to allow the camera to monitor the proteins within the container(s). One or more light sources (530) may be arranged to irradiate the labelled proteins, to enable the camerato capture details as desired. For example, if a fluorescently labelled protein is within a container, the light source may be configured to emit a wavelength of light that can activate the fluorescent tag, allowing the camera to capture the light emitted from the fluorescent tag. In some embodiments, the one or more lenses (522) may include a filter (524) for aiding in the detection of the protein tags. The camera and / or the light source may be coupled to a controller (540). The controller may include one or more processors (542), anon-transitory storage device (544), and memory (546). A display (550) may be coupled to the camera and / or the controller. The controller may be configured to control the light source and / or the camera, and the controller may be configured to store data (including one or more images I video) captured by the camera. In some embodiments, the controller is configured to display the image(s) or video captured by the camera. In some embodiments, the containers may be stationary relative to the camera. In some embodiments, the containers may move relative to the camera.

[0092] FIG. 9 provides a schematic summary' of the toolkit and strategy disclosed herein. The experimental components include bio-orthogonal fluorescent ligands, universal protein tags, such as HALOTAG® protein tags, can be used for controlled covalent labelling, and the use of ribosome homozygous tagging (RHT). As seen in FIG. 9, for resolving demographics, the basic concept is that pulse labelling begins the clock, following by a ligand washout and chase, where the chase moves the cells forward in time, allowing tracking over time. Spatial and compositional information can be attained via ligand fluorescence (e.g., using microscopy), and / or using the tracked proteins as input, with ligand-specific pulldown, the proteins can be fed to, e.g., data-independent acquisition (DIA) - mass spectrometry (MS), which can be used to map interactomes.

[0093] Experiments have been performed on human cells that validated the disclosed technology. The results demonstrate that genetic tagging of ribosomal proteins followed by conditional labeling with ligand allows for biochemical purification as well as confocal microscopy-based spatial characterization of human ribosomes as a function of age. This gave novel information on the spatial, compositional and functional histories of these macromolecular complexes and roles they may play in diseases like aging and ribosomopathies. The high degree of modularity of the disclosed process makes it particularly amenable for development into commercial kits with a universal protocol. The disclosed approach is superior to currently marketed products for characterizing protein composition and function in that it provides the additional dimensions of space and time.

[0094] Recording spatiotemporal compositional dynamics of nascent assemblies.

[0095] Referring to FIG. 10-11, it is shown how a U2OS RPS I 7-HaloTag can be used to immunopurify native ribosomes and characterize their steady-state composition, showing a comparison against a HaloTag-only control. This includes, e.g., western blots showing the ligand is effective for RPS18, RPL5 and RPL9, EIF4A1, and EEF1 A. Then, referring to FIG. 12, one can resort to nascent labeling of this ligand to purify nascent ribosomes and assess their compositional evolution over time.

[0096] FIGS. 13-17 showcase visualization of nascent ribosomes, their quantification, and proteomics data demonstrating the high quality of the purification possible using the disclosed approach. Here, cytoplasmic 40S maturation and function was considered using the basic process outlined in FIG. 13, including analyzing images captured, e.g., at 0, 2, 4, 8, 24, and additional images captured until the process reached a steady state. FIG. 14 shows the evolution of nascent 40S labelling over time, as a percent of the final steady state. FIG. 15 shows a graph of fold change vs adjusted p-value for comparing a RPS 17-HaloTag fusion (with a tetramethylrhodamine (TMR) dye) vs just the HaloTag(TMR). A preferred approach for the disclosed methodology utilizes this tetramethyrhodamine ligand to allow purification and imagine.

[0097] FIG. 16 shows a barchart of detected known sequences, using the disclosed approach, for cytoplasmic 40S immunoprecipitation (IP). FIG. 17 shows a visualization of the gene ontology for cytoplasmic 40S immunoprecipitation (IP).

[0098] FIGS. 18-23 demonstrate that the disclosed approach can indeed resolve the spatiotemporal compositional dynamics of ribosomes. Using nascent labelling to monitor the various processes, FIGS. 18-20 relate to assembly of ribosomes, while FIGS. 21A-23 relate to function of ribosomes. FIG. 18 illustrates the assembly of small subunits of ribosomal proteins over time while FIG. 19 illustrates the assembly of large subunits of ribosomal proteins over time. FIG. 20 illustrates change in assembly factors (including RRP12, RIOK1, PNO1, LTV1, NOB1, RIOK2, and BYSL) over time. FIGS. 21A-21B show expression of some subunits of eukaryotic initiation factor 3 (eIF3) over time. FIG. 22 shows expression of some subunits of eukaryotic initiation factor 4 (eIF4) over time. FIG. 23 shows expression of some subunits of eukaryotic elongation factors (eEFs) over time. Recording spatiotemporal compositional dynamics of pre-existing assemblies

[0099] Referring to FIG. 24, a basic process for tracking and recording spatiotemporal compositional dynamics of pre-existing assemblies can be seen. As shown, initially, the process involves labelling all steady-state intermediates, followed by a washout, followed by a block-chase period. Pre-existing ribosomal subunits in the nucleolus can be labeled, tracked in space and time, and compositional information over time can be obtained. The images shown the same portions of the nucleolus, tracked over time.

[0100] FIG. 25 shows core (e.g., fibrillar center) and rim (e.g., granular component) enrichment over time for large ribosomal subunit proteins. FIGS. 26-27 are charts tracking pre-60S ribosomal proteins (26) assembly factors (27) over time. FIG. 28 tracks nucleolar proteins over time. FIG. 29 shows the early depletion of certain nucleolar proteins, and when the depletion occurs. As seen, it is clear that the two proteins shown are generally not depleting at all around four hours into the process, whereupon both proteins suddenly get substantially depleted by hour six, and clearly almost entirely depleted by hour eight. Similarly, FIG. 30 shows how the disclosed pulse-chase strategy may be used to distinguish pre-existing and nascent small nucleolar ribonucleoproteins (snoRNP) complexes in HEK293 cells via Noppl40-HaloTag fusion. A JF646 pulse is used to label the pre-existing snoRNPs, while TMR is used during for the chase to label nascent snoRNPs.

Claims

What is claimed:

1. A method for characterizing compositional and functional histories of cellular proteins and their associated molecular complexes in space and time, comprising: forming fusion proteins (220) by fusing a universal genetic tag (222) to a plurality of endogenous proteins (210); performing at least one analysis selected from: tracking pre-existing complexes by: conditionally labelling the fusion protein (220) with a first ligand (224) by feeding cells with media (“cell feeding media”) containing the first ligand (224), the first ligand being a ligand with a developed immunopurification capability and a known imaging capability; allowing the labelled fusion proteins to bind the ligand covalently; removing the first ligand from the cell feeding media; and tracking pre-existing proteins over time; and / or tracking nascent complexes by: feeding cells with media containing a blocking ligand (230) that saturates all pre-existing complexes; washing the blocking ligand off the media; and feeding cells with media containing the first ligand to the cells, where only nascent protein that was not blocked with the non- fluorescent ligand can be bound; tracking and / or analyzing bound nascent protein over time.

2. The method of claim 1, further comprising recording a compositional and functional history of the plurality of endogenous proteins in space and time.

3. The method of claim 1 or 2, wherein a CRISPR-based technique is used to fuse genetic tags to the plurality of endogenous protein.

4. The method of any one of claims 1-3, wherein tracking is accomplished via live-cell confocal microscopy.

5. The method of any one of claims 1-4, wherein composition and function are determined by immunopurification followed by quantitative mass spectrometry and / or genomic analysis.

6. The method of any one of claims 1-5, further comprising: providing at least one of the fusion proteins to each of a plurality of containers; exposing each container to one or more chemical or biological agents; and identifying a chemical or biological agent that specifically targets an old or a young protein by performing the at least one analysis.

7. The method of any one of claims 1-6, wherein performing at least one analysis includes tracking pre-existing complexes.

8. The method of claim 7, wherein tracking pre-existing complexes includes: feeding cells with media containing the first ligand and allowing the fusion proteins to bind the first ligand covalently, washing out the first ligand to ensure no new proteins bind the ligand; and tracking the fusion proteins bound to the first ligand over time.

9. The method of any one of claims 1-8, wherein performing at least one analysis includes tracking nascent complexes.

10. The method of claim 9, wherein tracking nascent complexes includes: feeding cells with media containing a blocking ligand and allowing all pre-existing complexes to be saturated with the blocking ligand; washing off the blocking ligand; feeding the first ligand to the cells; and tracking nascent proteins bound to the first ligand.

11. The method of claim 1, wherein tracking is accomplished via live-cell confocal microscopy.

12. The method of claim 1, wherein composition and function are determined by immunopurification followed by quantitative mass spectrometry and / or genomic analysis.13 The method of claim 1, further comprising: providing at least one of the fusion proteins to each of a plurality of containers; exposing each container to one or more chemical or biological agents; and identifying a chemical or biological agent that specifically targets an old or a young protein by performing the at least one analysis.

14. The method of claim 1, wherein performing at least one analysis includes tracking preexisting complexes.

15. The method of claim 14, wherein tracking pre-existing complexes includes: feeding cells with media containing the first ligand and allowing the fusion proteins to bind the first ligand covalently, washing out the first ligand to ensure no new proteins bind the ligand; and tracking the fusion proteins bound to the first ligand over time.

16. The method of claim 1, wherein performing at least one analysis includes tracking nascent complexes.

17. The method of claim 16, wherein tracking nascent complexes includes: feeding cells with media containing a blocking ligand and allowing all pre-existing complexes to be saturated with the blocking ligand; washing off the blocking ligand; feeding the first ligand to the cells; and tracking nascent proteins bound to the first ligand.

18. A kit, comprising: a universal genetic tag; and a first ligand, the first ligand being a ligand with a developed immunopurification capability and a known imaging capability.

18. The kit of claim 18, wherein the first ligand is a halo tag ligand.

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