Mechanisms of cellular and intra-cellular scaling remain elusive. The use of Xenopus embryo extracts has become increasingly common to elucidate mechanisms of organelle size regulation. This method describes embryo extract preparation and a novel nuclear scaling assay through which mechanisms of nuclear size regulation can be identified.
A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally scales with the size of the cell, notably during embryogenesis when dramatic reductions in both cell and nuclear sizes occur. Mechanisms of nuclear size regulation are largely unknown and may be relevant to cancer where altered nuclear size is a key diagnostic and prognostic parameter. In vivo approaches to identifying nuclear size regulators are complicated by the essential and complex nature of nuclear function. The in vitro approach described here to study nuclear size control takes advantage of the normal reductions in nuclear size that occur during Xenopus laevis development. First, nuclei are assembled in X. laevis egg extract. Then, these nuclei are isolated and resuspended in cytoplasm from late stage embryos. After a 30 – 90 min incubation period, nuclear surface area decreases by 20 – 60%, providing a useful assay to identify cytoplasmic components present in late stage embryos that contribute to developmental nuclear size scaling. A major advantage of this approach is the relative facility with which the egg and embryo extracts can be biochemically manipulated, allowing for the identification of novel proteins and activities that regulate nuclear size. As with any in vitro approach, validation of results in an in vivo system is important, and microinjection of X. laevis embryos is particularly appropriate for these studies.
The sizes of cellular organelles typically scale with the size of the cell, and this has been perhaps best documented for the scaling of nuclear size with cell size1-10. This is particularly true during embryogenesis and cell differentiation, when dramatic reductions in both cell and nuclear size are often observed11,12. Furthermore, altered nuclear size is a key parameter in cancer diagnosis and prognosis13-17. Mechanisms that contribute to nuclear size regulation are largely unknown, in part due to the complexity and essential nature of nuclear structure and function. The method described here was developed as an in vitro assay for nuclear size scaling that is amenable to biochemical manipulation and elucidation of mechanisms of nuclear size regulation.
Xenopus laevis egg extract is a well-established system to recapitulate and study complex cellular processes in an in vitro context. These extracts have revealed new fundamental information about several cellular processes including the assembly and function of the mitotic spindle, endoplasmic reticulum, and nucleus18-22. One key advantage to the extract system is that X. laevis egg extracts represent a nearly undiluted cytoplasm whose composition can be easily altered, for instance through addition of recombinant proteins or immunodepletion. Furthermore, one is able to manipulate essential processes by employing treatments that might otherwise be lethal in an in vivo context. Modifications of the egg extract procedure allow for isolation of extracts from X. laevis embryos rather than eggs, and these embryo extracts are equally amenable to biochemical manipulation23. During X. laevis development, the single-cell fertilized embryo (~1 mm diameter) undergoes a series of twelve rapid cell divisions (stages 1 – 8) to generate several thousand 50 µm diameter and smaller cells, reaching a developmental stage termed the midblastula transition (MBT) or stage 824-26. The MBT is characterized by the onset of zygotic transcription, cell migration, asynchronous cell divisions, acquisition of gap phases, and establishment of nuclear steady-state sizes rather than continual nuclear expansion as in the pre-MBT embryo. From stage 4 to gastrulation (stages 10.5 – 12), the volume of individual nuclei decreases by more than 10-fold11.
Here, the goal is to identify mechanisms responsible for these reductions in nuclear size during developmental progression. The approach is to first assemble nuclei in X. laevis egg extract and to isolate those nuclei from the egg cytoplasm/extract. These nuclei are then resuspended in cytoplasm from late gastrula stage embryos. After an incubation period, the nuclei from egg extract become smaller in late stage embryo extract. We reasoned that this would be a useful assay for identifying cytoplasmic components present in late stage embryos that contribute to developmental nuclear size scaling. Using this assay, coupled with in vivo validation, we demonstrated that protein kinase C (PKC) contributes to developmental reductions in nuclear size in X. laevis23.
All Xenopus procedures and studies were conducted in compliance with the NRC Guide for the Care and Use of Laboratory Animals 8th edition. Protocols were approved by the University of Wyoming Institutional Animal Care and Use Committee (Assurance # A-3216-01).
1. Preparation of X. laevis Egg Extract (adapted from27,28)
2. Preparation of Demembranated X. laevis Sperm (adapted from29)
Note: The procedure volumes presented here are for up to 8 testes.
3. Nuclear Assembly
4. Preparation of X. laevis Embryo Extract
5. Nuclear Shrinking Assay and Immunofluorescence
Assembly of Nuclei in Egg Extract
The first steps of this protocol are to prepare X. laevis egg extract (Protocol 1) and demembranated sperm nuclei (Protocol 2). These reagents are then used to assemble nuclei de novo (Protocol 3). Figure 1 shows some representative data. Addition of calcium drives the meiotically arrested egg extract into interphase, and the cycloheximide keeps the extract arrested in interphase. By 30 – 45 min after initiation of the reaction, the initially thin S-shaped sperm nuclei should begin to thicken into slug-shaped chromatin masses. Successful formation of an intact nuclear envelope can be assessed by import and retention of a fluorescently labeled import cargo, such as GST-GFP-NLS (data not shown), or by rim staining for the nuclear pore complex using mAb414 that recognizes FG-repeat containing nucleoporins (Figure 1A). After nuclear assembly occurs, nuclear shape should become more rounded and the nuclear volume should increase as the nuclear envelope expands and nuclear proteins are imported (Figure 1B-C). Failure to observe formation of an intact nuclear envelope or nuclear growth indicates a poor quality egg extract. In this case, it is best to start again with a new batch of eggs.
Removal of Endogenous Nuclei from Embryo Extract
The next step is to prepare a late stage embryo extract (Protocol 4). After initial preparation of the extract and prior to removal of nuclei, it is best to prepare a Hoechst-stained squash of a small aliquot of the extract. Visualization of many small embryonic nuclei is a good indication of success in preparing the extract (Figure 2A). As endogenous embryonic nuclei can interfere with the downstream analysis, it is also important to check an aliquot of the extract after removing nuclei by dilution and centrifugation. Compared to the first squash, very few nuclei should be left in the extract (Figure 2B). If there are still many nuclei present, a second round of centrifugation is required.
Nuclear Shrinking Assay
Figure 3 shows representative results for the nuclear shrinking assay (Protocol 5). Egg extract nuclei resuspended in late stage embryo extract become smaller over time (Figure 3B). A good negative control for the assay is to incubate nuclei with embryo extract that has been heat inactivated (Figure 3A). As nuclei fail to shrink in heat inactivated extract, this provides some confidence that observed nuclear shrinking with untreated embryo extract is not a consequence of osmotic effects. The extent of nuclear size reductions observed varies depending on the particular batch of egg extract nuclei as well as the quality of the embryo extract. In our experience, nuclear shrinking is always observed (e.g., in more than 40 different embryo extracts), however it is important to repeat these experiments multiple times with different nuclei and extracts to address the inherent variability of the assay. Figure 4 shows a schematic of the entire protocol.
Figure 1. Nuclear Assembly Time Course in X. laevis Egg Extract. Nuclei were assembled de novo in X. laevis egg extract as described in Protocol 3. Aliquots from the same reaction were fixed and visualized by immunofluorescence using an antibody against the nuclear pore complex (NPC) (mAb414) at: A) 30 min, B) 60 min, and C) 90 min after the initiation of nuclear assembly. The immunofluorescence protocol is described in Protocol 5. The scale bar is 25 µm. Please click here to view a larger version of this figure.
Figure 2. Removal of Nuclei from X. laevis Embryo Extract. X. laevis embryo extract was prepared from stage 11.5 – 12 embryos as described in Protocol 4. A small aliquot of extract was fixed and stained with Hoechst: A) prior to removal of nuclei, and B) after removal of nuclei by centrifugation. The scale bar is 25 µm. Please click here to view a larger version of this figure.
Figure 3. Representative Data from the Nuclear Shrinking Assay. Nuclei were assembled de novo in X. laevis egg extract supplemented with recombinant GFP-LB3 (to visualize the nuclear lamina). As described in Protocol 5, egg extract nuclei were isolated and resuspended in stage 11.5 – 12 embryo extract from which endogenous embryonic nuclei had been removed. Live time-lapse imaging was performed at 30 sec intervals for 90 min. Figure panels show images acquired at 6 min intervals for nuclei incubated in: A) late stage embryo extract (LEE), and B) heat inactivated (HI) embryo extract. Images from the time courses proceed from left-to-right. The scale bar is 25 µm. C) Nuclear shrinking data from 46 different egg and embryo extracts. Bars show the means for > 240 nuclei. Error bars are standard deviation. Please click here to view a larger version of this figure.
Figure 4. Schematic of the Nuclear Shrinking Assay. Each colored box represents one protocol. Red indicates preparation of X. laevis egg extract (Protocol 1). Blue indicates preparation of demembranated X. laevis sperm (Protocol 2). Pink indicates de novo nuclear assembly in egg extract (Protocol 3). Green indicates preparation of X. laevis embryo extract (Protocol 4). Purple indicates the nuclear shrinking assay protocol and immunofluorescence (Protocol 5). Portions of this figure have been reused from23. Please click here to view a larger version of this figure.
Buffer name | Buffer composition |
Marc's Modified Ringers (MMR), 1/3x | 33 mM NaCl, 0.7 mM KCl, 0.3 mM MgSO4, 0.7 mM CaCl2, 1.7 mM HEPES, pH 7.4 |
Extract buffer (XB), 10x | 1 M KCl, 1 mM CaCl2, 10 mM MgCl2, 500 mM sucrose, 100 mM HEPES, pH 7.8 (pH adjusted with 10 N KOH, stored at 4 °C) |
Buffer B | 4% Cysteine dissolved in 0.8x XB (prepared with cold ddH2O, pH adjusted to 7.8 with 10 N NaOH) |
Buffer C | Mix 100 m of 10x XB with 900 ml of cold ddH2O |
Buffer D | Buffer C plus 5 mM EGTA and 800 µM MgCl2 |
LPC stock, 1,000x | 10 mg/ml each of leupeptin, pepstatin, and chymostatin dissolved in DMSO (stored in aliquots at -20 °C) |
Buffer E | 100 ml Buffer D plus 100 µl LPC stock |
Energy mix, 50x | 190 mM creatine phosphate disodium, 25 mM ATP disodium salt, 25 mM magnesium chloride |
Buffer T | 15 mM PIPES, 15 mM NaCl, 5 mM EDTA, 7 mM MgCl2, 80 mM KCl, 0.2 M sucrose, pH 7.4 (filter sterilize and stored at 4 °C) |
Buffer S | 20 mM maltose and 0.05% lysolecithin prepared in Buffer T |
Buffer R | Buffer T plus 3% bovine serum albumin |
Calcium stock solution, 20x | 10 mM CaCl2, 0.1 M KCl, 1 mM MgCl2 (stored at -20 °C) |
Nucleus fix | 125 µl 2 M sucrose, 12.5 µl 1 M HEPES pH 7.9, 250 µl 37% formaldehyde, 112 µl ddH2O, 0.5 µl 10 mg/ml Hoechst (stored at room temperature for up to two weeks) |
Modified Barth's Saline (MBS), 10x | 880 mM NaCl, 10 mM KCl, 50 mM HEPES, 25 mM NaHCO3, pH 7.8 |
High Salt MBS | 1x MBS plus 0.7 mM CaCl2 and 20 mM NaCl |
Egg lysis buffer, ELB | 250 mM sucrose, 50 mM KCl, 2.5 mM MgCl2, 10 mM HEPES, pH 7.8 |
Nuclear cushion buffer | 1x XB, 0.2 M sucrose, 25% glycerol (filter sterilized and stored at 4 °C) |
Table 1. Buffers. Compositions of all buffers mentioned in this protocol are described in this table.
Here is presented a novel method to study mechanisms of nuclear size regulation during X. laevis development. Developmental progression is associated with dramatic changes in cell physiology, metabolism, division rates, and migration, as well as alterations in the sizes of cells and intracellular structures. These varied processes are complex and essential, so it is difficult to study just one of these aspects of development in an in vivo setting. The X. laevis embryo extract and nuclear shrinking assay described here allow for the study of size regulatory mechanisms in an in vitro context, circumventing the complex signals and physical constraints of the in vivo system. Furthermore, the open nature of the extract allows for its facile biochemical manipulation.
Troubleshooting some steps of the protocol may be necessary. Problems with nuclear assembly and growth can result from poor quality eggs, incomplete demembranation of sperm nuclei, use of sperm nuclei at too high of a concentration, or incubating nuclear assembly reactions at too low of a temperature. Removal of endogenous embryonic nuclei from late embryo extract can be difficult due to the small size of these nuclei. This step in the protocol may require more than one round of centrifugation or the use of alternate rotors to completely remove all nuclei. Failure of nuclei to become smaller in the shrinking assay may be due to poor quality embryos, starting with nuclei that are too small, or too low of an incubation temperature. Poor quality eggs and embryos are indicated by a large proportion of white puffy activated eggs, unequal distribution of yolk and pigment granules, and eggs laid in strings. Spinning down fixed nuclei onto coverslips occasionally causes fragmentation of the nuclei, which can be avoided by decreasing the centrifugation speed or increasing the glycerol and sucrose concentrations in the nucleus cushion buffer. Troubleshooting antibody selection and dilution for immunofluorescence of nuclei is also often necessary. Critical steps within these protocols include: using high quality eggs and embryos, removing activated and lysed eggs and embryos, ensuring sperm nuclei are fully demembranated and appropriately diluted, monitoring nuclear assembly and growth, correctly staging embryos, completely removing endogenous embryonic nuclei from late stage embryo extracts, centrifuging fixed nuclei onto glass coverslips, and immunofluorescent staining of nuclei for visualization and quantification.
Nuclear size differences have long been noted between different species, cell types, disease states, and stages of embryonic development, indicating that nuclear size is tightly regulated1-10. Cancer of the prostate, breast, and ovary, to name a few, are all diagnosed and staged based on graded increases in nuclear size14,30-32. It is unclear to what extent changes in nuclear size are a cause or consequence of disease, and whether normal scaling of nuclear size contributes to proper development. Answering these functional questions necessitates an understanding of the mechanisms that control nuclear size. The assay presented here provides one approach to identifying novel pathways and proteins that regulate nuclear size, with potential relevance to cancer and development.
The Xenopus egg and embryo extract systems provide particularly powerful approaches to investigate mechanisms of organelle size regulation. While Xenopus extracts have been used for decades to study various aspects of cell cycle regulation and organelle assembly and dynamics, these extracts have more recently provided insights into size regulation of the nucleus3,23,33, mitotic spindle34-36, and chromosomes37-39. One of the major advantages of working with X. laevis embryo extracts is that they can be isolated from different stages of development, representing cytoplasm from differently sized cells containing nuclei of differing sizes. The method for investigating nuclear re-sizing presented here is physiologically relevant and offers a simplified system with which to identify mechanisms of nuclear size regulation. One complication of studying size regulation in vivo is that it is difficult to distinguish effects of spatial constraints and volume-limited components on size versus developmentally regulated changes in the expression or activities of size-controlling factors40. The approach described here focuses on developmentally controlled nuclear size regulators, using a robust nuclear re-sizing assay that is amenable to biochemical dissection.
Using the methods presented here, we identified protein kinase C (PKC) as a novel regulator of nuclear size. Furthermore, we showed that PKC activity and nuclear localization increase during X. laevis development23,41. A key limitation to the nuclear shrinking assay is that it is performed entirely in vitro. While Xenopus extract systems provide a simplified platform to discover new factors that regulate size, it is important that these results are validated in vivo. There are a variety of different approaches to corroborate physiological significance. X. laevis embryos can be microinjected with mRNA or morpholinos to alter the levels of nuclear scaling factors identified in extract, and then the effects on nuclear size can be quantified. Treatment of embryos with cell-permeable small molecule inhibitors or activators is also possible. For instance, we observed nuclear shrinking in live embryos treated with a phorbol ester, and this shrinking occurred within interphase without a requirement for nuclear envelope breakdown and passage through mitosis23. Another approach is to alter the levels of nuclear scaling factors in tissue culture cells to assess conservation of nuclear size control mechanisms outside of Xenopus.
What are the functional implications of altered nuclear size? One possibility is that nuclear size influences chromosome and chromatin organization, thereby altering gene expression. This idea can be directly tested as mechanisms of nuclear size regulation are further elucidated. Interestingly, PKC signaling and nuclear size are both routinely deregulated in cancer cells, often representing more aggressive metastatic disease13,14,42,43. Whether there is a direct relationship between deregulated PKC signaling and altered nuclear size in cancer remains to be shown. There is still much to be learned about nuclear size regulation, and the assay presented here, or variants of it, may still yield new information about mechanisms of nuclear size control. Furthermore, this assay could easily be adapted for the study of scaling mechanisms of other organelles and intracellular structures such as the mitotic spindle, endoplasmic reticulum, chromosomes, and Golgi, to name a few.
The authors have nothing to disclose.
Members of the Levy and Gatlin labs as well as colleagues in the Department of Molecular Biology offered helpful advice and discussions. Rebecca Heald provided support in the early stages of developing this protocol. This work was supported by the NIH/NIGMS (R15GM106318) and the American Cancer Society (RSG-15-035-01-DDC).
Alexa Fluor 568 Donkey anti-mouse IgG | Molecular Probes | A10037 | |
ATP disodium salt | Sigma Aldrich | A2383 | |
Benzocaine | Sigma Aldrich | E1501 | |
Bovine Serum Albumin | Sigma Aldrich | A3059 | |
CaCl2 | Sigma Aldrich | C3306 | |
Centrifuge | Beckman | J2-21M | |
Centrifuge rotor | Beckman | JS 13.1 | |
chymostatin | Sigma Aldrich | C7268 | |
creatine phosphate disodium | Calbiochem | 2380 | |
cycloheximide | Sigma Aldrich | C6255 | |
cytochalasin D | Sigma Aldrich | C8273 | |
disposable wipes (kimwipes) | Sigma Aldrich | Z188956 | |
L-cysteine | Sigma Aldrich | W326306 | |
EGTA | Sigma Aldrich | E4378 | |
Formaldehyde | Sigma Aldrich | F8775 | |
Glass crystallizing dish (150×75 mm) | VWR | 89090-662 | |
Glycerol | Macron | 5094-16 | |
HEPES | Sigma Aldrich | H4034 | |
Hoechst – bisBenzimide H 33342 trihydrochloride | Sigma Aldrich | B2261 | |
HCG – Human Chorionic Gonadotropin | Prospec | hor-250-c | |
L15 Media | Sigma Aldrich | L4386 | |
leupeptin | Sigma Aldrich | L2884 | |
Lysolecithin | Sigma Aldrich | L1381 | |
mAb414 | Abcam | ab24609 | |
MgCl2 | EMD | MX0045-2 | |
MgSO4 | Sigma Aldrich | M9397 | |
Maltose | Sigma Aldrich | M5885 | |
NP40 | BDH | 56009 | |
Paraformaldehyde | Electron Microscopy Sciences | 15710 | |
Penicillin + Streptomycin | Sigma Aldrich | Pp0781 | |
pepstatin | Sigma Aldrich | P5318 | |
PIPES | Sigma Aldrich | P6757 | |
Plastic paraffin film (parafilm) | Sigma Aldrich | P7793 | |
KCl | Sigma Aldrich | P9541 | |
KH2PO4 | Mallinckrodt | 70100 | |
KOH | Baker | 5 3140 | |
PMSG – Pregnant Mare Serum Gonadotropin | Prospec | hor-272-a | |
NaCl | Sigma Aldrich | S3014 | |
NaHCO3 | Fisher | BP328 | |
NaHPO4 | EMD | SX0720-1 | |
NaOH | EMD | SX0590 | |
Pestle | Thomas Scientific | 3411D56 | |
Round bottom glass tubes, 15 ml | Corex | 8441 | |
Secondary antibody (Alexa Fluor 568 donkey anti-mouse IgG) | ThermoFisher | A10037 | |
sucrose | Calbiochem | 8550 | |
thermal cycler | Bio-Rad | T100 | |
Ultracentrifuge | Beckman | L8-80M | |
Ultracentrifuge rotor | Beckman | SW 50.1 | |
Vectashield (anti-fade mounting medium) | Vector | H-1000 |