Histochem Cell Biol DOI 10.1007/s00418-013-1167-9

Original Paper

Probing the stiffness of isolated nucleoli by atomic force microscopy Emilie Louvet · Aiko Yoshida · Masahiro Kumeta · Kunio Takeyasu 

Accepted: 24 October 2013 © Springer-Verlag Berlin Heidelberg 2013

Abstract  In eukaryotic cells, ribosome biogenesis occurs in the nucleolus, a membraneless nuclear compartment. Noticeably, the nucleolus is also involved in several nuclear functions, such as cell cycle regulation, non-ribosomal ribonucleoprotein complex assembly, aggresome formation and some virus assembly. The most intriguing question about the nucleolus is how such dynamics processes can occur in such a compact compartment. We hypothesized that its structure may be rather flexible. To investigate this, we used atomic force microscopy (AFM) on isolated nucleoli. Surface topography imaging revealed the beaded structure of the nucleolar surface. With the AFM’s ability to measure forces, we were able to determine the stiffness of isolated nucleoli. We could establish that the nucleolar stiffness varies upon drastic morphological changes induced by transcription and proteasome inhibition. Furthermore, upon ribosomal proteins and LaminB1 knockdowns, the nucleolar stiffness was increased. This led us to propose a model where the nucleolus has steady-state stiffness dependent on ribosome biogenesis activity and requires LaminB1 for its flexibility. Keywords Nucleolus · AFM · Stiffness · Nucleolar structure E. Louvet (*) · A. Yoshida · M. Kumeta · K. Takeyasu  Graduate School of Biostudies, Kyoto University, Kyoto 606‑8501, Japan e-mail: [email protected] A. Yoshida e-mail: [email protected]‑u.ac.jp M. Kumeta e-mail: [email protected]‑u.ac.jp K. Takeyasu e-mail: [email protected]‑u.ac.jp

Abbreviations Pol. I RNA polymerase I rDNA Ribosomal DNA FC Fibrillar center DFC Dense fibrillar component GC Granular component AFM Atomic force microscopy rRNA Ribosomal RNA YM Young’s modulus Pa Pascal PLL Poly-l-lysine RT Room temperature

Introduction The nucleolus is a nuclear body which main function is ribosome biogenesis. The ribosomal DNA (rDNA) is transcribed by the RNA polymerase I (pol. I), giving rise to pre-rRNA that is processed in a sequential manner in order to produce the 18S, 5.8S and 28S rRNA. These events can be seen as a vectorial process in which the earlier events take place inside the nucleolus and the later events take place in its outer shell, to be continued outside of this body. Indeed, the nucleolus is commonly described as being composed of three compartments: the fibrillar center (FC) contains the ribosomal genes and transcription machineries. The FC is embedded in the dense fibrillar component (DFC). The transcription of rDNA takes place at the junction of the FC and DFC. The DFC is the site of early rRNA processing and is itself embedded in a thicker shell: the granular component (GC) where the late rRNA processing events occur (Derenzini et al. 2006; Hernandez-Verdun 2006a; Raska et al. 2006). This well-established compartmentation is, however, challenged by recent studies. The nucleolus is involved

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in several nuclear functions, such as cell cycle regulation, non-ribosomal ribonucleoprotein complex assembly, aggresome formation and some virus assembly (Hiscox 2007; Jacobson and Pederson 1998; Krüger and Scheer 2010; Latonen et al. 2011; Politz et al. 2000; Shou et al. 1999; Visintin et al. 1999; Weber et al. 1999). Interestingly, several of these functions occur in the GC or take place in compartments different from those dedicated to the ribosomal subunits biogenesis (Krüger and Scheer 2010; Latonen et al. 2011; Politz et al. 2000). So, over the last years, it has not only been demonstrated that the nucleolus is a multifunctional nuclear body, but it has also become evident that its structure is more complex than the tripartite structure commonly described (Lamaye et al. 2011). The structural data of the nucleolus and some biophysical parameters, such as the mobility of nucleolar proteins, have mostly been obtained by far-field microscopy techniques such as electron and fluorescence microscopy. The electron microscopy methods have mainly enabled the fine characterization of the structural compartmentation of the nucleolus (Cheutin et al. 2003; Cmarko et al. 2000; Derenzini et al. 2006; Hernandez-Verdun 2006b; Lamaye et al. 2011; Raska et al. 2006; Smetana et al. 1970a, b). The fluorescence microscopy has also contributed to structural characterization through the mapping of sites in the GC, where non-ribosome biogenesis-related functions occur (Krüger and Scheer 2010; Latonen et al. 2011; Politz et al. 2000), and the discovery of a new compartment in the FC (Hutten et al. 2011). Moreover, the fluorescence methods have allowed for the characterization of the mobility of nucleolar proteins (Chen and Huang 2001; Dundr et al. 2002; Louvet et al. 2005; Shav-Tal et al. 2005) and for the characterization of dynamic structural changes in the nucleolus during the cell cycle and upon drug treatment (Dundr et al. 2000; Leung et al. 2004; Louvet et al. 2005; Savino et al. 2001). Since its discovery by Fontana in 1781, the nucleolus is depicted as a dense and compact compartment, but its “compactness” is now in question (Pederson 2010). Nucleoli are only twice as dense as the nucleoplasm (Handwerger et al. 2005). The diffusion of ovalbumin, both in the nucleoplasm and in the nucleolus, is similar (Speil and Kubitscheck 2009). So, the lack of penetration of free molecules in the nucleolus is due to the lack of available space (Speil and Kubitscheck 2009). Considering the amount of ribosomal subunits that need to be produced, the multiple functions performed by the nucleolus, its complex structure and the morphological changes that it encounters, one can question how such dynamic processes can occur in such a “compact” compartment. We hypothesized that the nucleolus is not simply a compact structure, but it is a flexible structure. In order to investigate its structure and flexibility, we used atomic force microscopy (AFM), with the different measurements

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Histochem Cell Biol

it offers. Indeed, the scanning probe microscopy techniques, such as AFM, offer the possibility, by sensing the sample, to obtain topography information and biophysical parameters (Binnig et al. 1986; Binnig and Rohrer 1982; Hansma et al. 1988). As a model, we worked on HeLa cells because the nucleolar isolation protocol is well established on this cell line. We imaged the surface topography of isolated nucleoli: the nucleolar surface exhibits a beaded structure. By measuring the nucleolar stiffness, we show that the nucleolus is a flexible structure that varies upon morphological and functional modification. We also show that the nucleolar stiffness depends on LaminB1, which suggests the contribution of nuclear scaffold proteins to the regulation of nucleolar flexibility.

Results Nucleolar structure and permeability after isolation Nucleoli were isolated from HeLa cells using a protocol adapted from previously established methods (Fig. 1a) (Andersen et al. 2002; Hacot et al. 2010; Scherl et al. 2002) and spotted on coated coverslips for immunofluorescence. Their axial sizes have been measured, and they ranged from 2.7 to 7.5 μm (Fig. 1b). Their thickness was measured by acquiring confocal stacks and comprised between 2.5 and 4  μm (not shown). The recorded sizes are in accordance with those found in the literature. The DFC was detected by immunofluorescence with an antibody against fibrillarin, and the GC was detected with B23 and nucleolin (Fig. 1c). The distribution of the markers showed that the structural organization of the isolated nucleoli is similar to those of intact cells. To further verify the purity of the nucleoli fraction, the protein composition of total cell, nuclei and nucleoli extracts was analyzed by loading the same amount of protein per lane on SDS–PAGE. The nucleoli extract exhibits a greater amount of proteins migrating at 37 kDa, molecular weight that corresponds to B23 (Fig. 1d). Western blotting analysis revealed indeed an increase in B23 in nucleolar fraction. An important decrease in LaminB1 was observed in the nucleolar fraction in comparison with the nuclear fraction, and GAPDH was not found in either the nuclear or the nucleolar fractions (Fig. 1e). All these data demonstrate that the prototypical organization of the nucleolus was maintained through the isolation procedure. The nucleolus is often depicted as a compact structure. In order to assess the permeability of the nucleolus, isolated nucleoli from a GFP–B23 stably expressing cell line were incubated with three different sizes of dextran: 10, 40 and 70 kDa coupled to tetramethylrhodamine or rhodamine. The nucleoli were not fixed and required a buffer that ensured the preservation of their structure as well as no loss

Histochem Cell Biol Fig. 1  Nucleolar isolation. a Nucleolar isolation protocol. Yellow, green and pink represent the fibrillar center (FC), dense fibrillar component (DFC) and the granular component (GC), respectively. b Nucleolin labeling used for nucleoli detection. Short axis measurement (red) exhibits a size range from 2.76 to 3.94 μm and long axis measurement (white) from 4.71 to 7.49 μm. Scale bar 5 μm. c B23 and nucleolin (Nu) (GC marker) and fibrillarin (Fib) (DFC marker) labeling. Scale bars 5 μm. d Coomassie staining of 12 % SDS–PAGE gel. Ten micrograms of total cell extract (C), nuclear extract (N) and nucleolar extract (Nu) was loaded per lane. The asterisk shows a band at approximately 37 kDa appearing in the nucleolar extract that corresponds to the molecular weight of B23. Sizes of molecular weight markers are indicated on the left. e Western blot performed on the same extracts to reveal B23, LaminB1 and GAPDH proteins

of proteins (Handwerger et al. 2005). The nucleoli were therefore incubated in a sucrose-containing buffer. Both the 10-kDa and the 40-kDa dextran penetrated the nucleoli and were homogenously distributed in the GC (Fig. 2). When nucleoli were incubated with the 70-kDa dextran, its penetration was not as homogeneously spread in the nucleoli as the 10- and 40-kDa dextran (Fig. 2). Indeed, a stronger labeling of the periphery of the nucleolus was detected. This labeling did overlay with the intense Hoechst staining of its periphery that corresponds to the heterochromatin shell surrounding the nucleoli, as previously shown with multiple methods (Ferreira et al. 1997; Németh et al. 2010; Sadoni et al. 1999; Smetana et al. 1968; van Koningsbruggen et al. 2010). As the 70-kDa dextran was partly

retained in the heterochromatin shell of the nucleolus, we set out to image the surface of isolated nucleoli by AFM in order to reveal the surface topography of the nucleolus. Nucleoli were imaged in air and liquid conditions, low versus high resolution, respectively. We observed 28 nucleoli in air, of which the surface topography revealed a beaded structure. The diameter of the beads was measured, and the average diameter was 271 ± 126 nm (n  = 543, ±SD) (Fig. 3a). To increase the resolution and decipher the structure of the identified beads, imaging in liquid was performed (Fig. 3b, c). We used BIXAM, a fastscanning AFM coupled to an inverted fluorescence microscope (Suzuki et al. 2013). Nucleoli were isolated from a GFP–UBF stably expressing cell line in order to select

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Histochem Cell Biol

Fig. 2  Isolated nucleoli permeability. Nucleoli were isolated from a GFP–B23 stably expressing cell line in order to follow the incorporation of dextran coupled to tetramethylrhodamine (10 and 40 kDa) and rhodamine (70 kDa). The DNA surrounding the nucleoli was revealed

by Hoechst incorporation. The merge between the dextran (red) and DNA (blue) shows that the 10- and 40-kDa dextran penetrated homogeneously the nucleoli, whereas the 70-kDa accumulated in the DNA surrounding the nucleoli. Scale bars 5 μm

nucleoli by fluorescence for AFM imaging (Fig. 3b). This imaging method allowed us to determine that the beads of 271 nm identified in air were made of smaller beads with an average diameter of 57 ± 25 nm (n = 43, ±SD) (Fig. 3c, examples of small beads are shown by white arrow heads).

The YM of isolated nucleoli was in the range of 30 × 103 Pa (Fig. 4h and see Table 1 for raw data and number of measurements). The YM of polystyrene beads with a diameter of ~5 μm (same size range as nucleoli) and of known YM was measured (Fig. 4c, f). The calculated YM of the polystyrene beads comprised between 9.68 × 108 and 8.95 × 109 Pascal (Pa) (average 3.63 × 109 Pa), which is concordant with reported values of polystyrene YM, i.e., between 3 and 3.5 × 109 Pa (http://www.engineeringtool box.com/young-modulus-d_417.html) (Fig. 4f). The YM of isolated nucleoli was next measured using a cantilever coupled to a sphere of 2 μm diameter in order to increase the surface of contact between the sample and the cantilever (Fig. 4g). The YM of the isolated nucleoli was in the range of 36 × 103 Pa (Fig. 4h and see Table 1 for raw data and number of measurements). Using two different types of cantilever, the calculated YM of the isolated nucleoli was in the same range, validating the measurements. However, one should consider that the YM of the nucleoli, due to the heterogeneity of the sample, is not an absolute value. Therefore, in the subsequent experiments, relative change of YM between the control and treated samples was analyzed.

Structural organization and stiffness of isolated nucleoli Nucleoli were seeded on poly-l-lysine-coated glass bottom dishes in sucrose-containing buffer (the same buffer as the one used for the dextran penetration assay) (Fig. 4a). The center of a nucleolus is targeted in order to apply force with the pyramidal-shaped tip of the cantilever. The tip of the cantilever is indicated on the cantilever as the intersection of a cross (Fig. 4b). The application of a 2 nN force was needed, on the nucleoli, in order to record force curves with an appropriate signal-to-noise ratio, and to obtain enough indentation in order to calculate the Young’s modulus (YM) (Fig. 4e). In order to avoid substrate effect from the glass, no more than 20–30 % of the total height of the sample was indented (Domke and Radmacher 1998). After force measurements, nucleoli were still intact (Fig. 4d).

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Histochem Cell Biol Fig. 3  Nucleolus surface exhibits a beaded structure. a AFM surface topography of isolated nucleoli in air. The nucleoli were imaged in fields of 5 × 5 μm. The size of the beads was measured and plotted as a histogram: 543 beads were measured on 28 nucleoli. The average size is 271 ± 126 nm. b and c AFM surface topography of isolated nucleoli in liquid. b Under BIXAM, phase contrast and fluorescence allowed for the selection of the nucleolus to be imaged. The cantilever visualized by phase contrast was targeted to the nucleolus of interest. Scale bar 5 μm. c Surface topography of the edge of a nucleolus (scan size 2 × 1.5 μm, scale bar 200 nm), 3D view zoom revealing the smaller bead composition, white arrow heads show examples of small beads (1 × 0.75 μm, scale bar 200 nm) and histogram plotting the size of the beads. The average size is 57 ± 25 nm (43 beads measured on 1 nucleolus)

In order to test whether the nucleoli stiffness varied, cells were treated with drugs that dramatically alter the nucleolar structure. Upon actinomycin D (AMD) treatment, used at a concentration to inhibit pol. I transcription, nucleolar compartments are segregated: FC and DFC are located on the edges of the nucleolus, forming the so-called nucleolar caps, and the GC formed a central body (Fig. 5a) (Shav-Tal et al. 2005). Upon MG132 treatment, a proteasome inhibitor, an aggresome is formed in the GC. This aggresome formation does not inhibit the nucleolar function, and the nucleolus compartments are rearranged around this structure (KarniSchmidt et al. 2008; Krüger and Scheer 2010; Latonen et al. 2011). Indeed, the GC takes a donut or croissant shape around the aggresome, and the FCs and DFCs are also surrounding the aggresome (Fig. 5b). The aggresomes contain numerous proteins, e.g., cyclins, transcription factors, proteasome core proteins and PML (Latonen 2011). Indeed, we could observe the accumulation of PML (Fig. 5c). Cells were treated with either AMD or MG132, and nucleoli were isolated and prepared for force measurement. As the YM of biological sample, due to its heterogeneity,

is not an absolute value (Radmacher 1997; Yokokawa et al. 2008), the YM of drug-treated samples (AMD and MG132) was always compared against untreated nucleoli (control) in order to analyze whether relative YM changes could be observed between the control and treated samples. The average histogram of three independent experiments is shown in Fig. 5d. The measured stiffness of the control nucleoli was about 30 kPa, while those treated with AMD were softer, at 26 kPa, and those treated with MG132 were stiffer, at 36 kPa (see Table 2 for raw data and number of measurements). These results show that the stiffness of the nucleolus varies upon drastic structural modification and that it is possible to measure this difference. Nucleolar stiffness changes upon alteration in ribosomal biogenesis or LaminB1 down regulation In order to test whether the nucleolar stiffness varied upon ribosomal biogenesis alteration, we chose to target rRNA processing. One constraint was the necessity to alter these processes without losing the ability to isolate the nucleolus,

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i.e., without losing a nucleolar structure that maintains the three components. It was therefore impossible to use drugs traditionally used to inhibit rRNA processing such as the DRB and roscovitine that unravel the nucleolus (Louvet et al. 2005, 2006; Sirri et al. 2002). Thus, we chose to knock down the expression of ribosomal proteins by siRNA. The siRNA

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targets were chosen so as to knock down a ribosomal protein involved in each pathway: RPS6 (small subunit generation, 18S pathway) and RPL11 (large subunit generation, 28S pathway) because of their detailed characterization in rRNA processing (Fumagalli et al. 2009; Robledo et al. 2008). There is a correlation between the function and the structure of the

Histochem Cell Biol

◂ Fig. 4  Nucleolar stiffness measurement. a Nucleoli are seeded on a

glass bottom dish coated with PLL. The cantilever (gray) is targeted on the middle of the nucleolus, and 2 nN force is applied in order to record one force curve. b Top view of the cantilever over isolated nucleoli. The tip of the cantilever is represented by the cross-intersection indicated by the black arrow. Scale bar 10 μm. c Cantilever and 5-μm-diameter polystyrene beads. Scale bar 10 μm. d In order to calculate the YM of a nucleolus, ten force curves are measured. The first picture shows the targeted nucleolus (white arrow), the second picture shows the tip engaged on this nucleolus (white arrow), and the third picture shows the same nucleolus after ten force curve measurements. The nucleolus is still intact (white arrow). e Example of a force curve obtained after indentation of a nucleolus. The blue curve corresponds to the indentation curve and is fitted to the Hertz– Sneddon model in order to determine the best YM fit (gray curve). The calculated YM (Pa) is indicated in green. The red curve corresponds to the retraction curve. f Example of a force curve measured on a polystyrene bead. Note the difference in the slope of the curve and the calculated YM (green annotation). g Example of a force curve measured on a nucleolus with a spherical indenter. Note that the calculated YM is in the same range than the calculated YM after indentation with a pyramidal cantilever (green annotation). h Histogram representing the averaged calculated YM of isolated nucleoli with the pyramidal indenter (pyramidal, blue bar) and with the spherical indenter ( spherical,red bar). The average calculated YM of isolated nucleoli was 30 kPa with the pyramidal indenter, and it was of 36 kPa with the spherical indenter. See Table 1 for raw data and number of measurements

nucleolus; therefore, it was important to characterize how the nucleolar morphology is affected after knockdown of RPS6 and RPL11 (Fig. 6). To this end, a cell line permanently expressing GFP–B23 was used to follow the GC, and the DFC was labeled with an antibody targeting fibrillarin. After knockdown of RPS6, the nucleoli’s shape was more circular (shown by the GC) than the control nucleolus. The nucleolus aspect ratio was 1.273 (n = 170) versus 1.446 for the untreated cells (n  = 117), the value 1 being a perfect circle. The DFC was not as spread out as in control nucleoli but had a concentric shape. After RPL11 knockdown, the GC was elongated and its border was irregular. The aspect ratio after RPL11 knockdown was 1,557 (n  = 147). The DFC was more spread out in the nucleoli, forming a kind of necklace structure following the GC shape (Fig. 6). After knockdown of GAPDH and negative control (NegC), there was no visible alteration or morphological modification of the nucleolar structure (Fig. 6) (aspect ratios were similar to the control value with 1.428, n = 113, and 1.363, n  = 119, respectively). Although the RPS6 and RPL11 knockdowns have a slightly modified structure, it is only a mild modification in comparison with those seen with drug treatments. The knockdown efficiencies were measured by Western blot (Fig. 7a). The intensities of the bands were quantified and normalized with ß-actin signal, and the knockdown efficiencies were estimated to be 68 % for RPS6, 45 % for RPL11 and 55 % for GAPDH (Fig. 7b–d). The stiffness measurement of isolated nucleoli showed that knockdown of RPS6 and RPL11 increased the stiffness of the nucleoli, to be, respectively, 30 and 15 % higher than

Table 1  Raw data of isolated nucleoli Young's moduli using a pyramidal versus a spherical indenter

YM (Pa) Nucleoli number Force curves number

Pyramidal

Spherical

30,962 194

36,718 29

1,473

212

control (Fig. 8 and see Table 3 for raw data and number of measurements). The two control siRNAs, siGAPDH and siNegC, did not affect the YMs. Therefore, the nucleolar stiffness is increased upon ribosome biogenesis alteration. Lamins have been shown to be present not only in the lamina, but throughout the nucleus, and function as regulators of RNA transcription (Hozák et al. 1995; Tang et al. 2008). We knocked down LaminB1 in a GFP–B23 stably expressing cell line in order to visualize its effect on the nucleolus shape, through the distribution of B23 (Fig. 9a, b). The decrease in LaminB1 was of 65 %, as revealed by Western blot (Fig. 9c). As previously reported (Martin et al. 2009; Tang et al. 2008), following LaminB1 knockdown, nucleoli exhibited a stretched structure; the DFC formed a grapelike structure, as opposed to the typical foci distribution observed in control nucleoli (Fig. 9b). We also noticed that B23 diffused to the nucleoplasm in some cells (Fig. 9a). The stiffness measurements of control versus LaminB1 knockdown nucleoli showed that the YM was higher after LaminB1 knockdown: the calculated YM was about 64 kPa for the knockdown, and about 52 kPa for the control (Fig. 9d and see Table 4). Therefore, our experiment demonstrates that LaminB1 is important for the nucleolus flexibility as the stiffness increases upon its depletion.

Discussion This study investigates for the first time the structure and the stiffness of isolated nucleoli by AFM. We revealed by surface topography imaging the beaded structure of the heterochromatin shell surrounding the nucleolus. We were able to measure the stiffness of the nucleolus and to determine that it varies upon structural rearrangement, upon alteration in the ribosome biogenesis and upon decreases in LaminB1 expression. As there is no precedent for this type of study, our work is generating more questions than answers, but it paves the way for new means to investigate the regulation and maintenance of the nucleolar structure. Heterochromatin: a nucleolar barrier? In order to investigate the nucleolar permeability, we tested the penetration of 10-, 40- and 70-kDa dextran molecules

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Histochem Cell Biol

Fig. 5  Nucleolar stiffness variation upon structural changes. a B23 (red) and fibrillarin (Fib) (green) labeling on an isolated nucleolus after AMD treatment. b B23 (red) and Fib (green) labeling on an isolated nucleolus after MG132 treatment. c UBF (red) and PML (green) labeling on an isolated nucleolus after MG132 treatment. Scale bars 5 μm. d Histogram representing the averaged YM of isolated nucleoli of three independent experiments from untreated cells (control, blue bar), AMD-treated (red bar) and MG132-treated cells (green bar). The YM values were 26 and 36 kPa after, respectively, AMD treatment and MG132 treatment, whereas the control cells exhibit an YM of 30 kPa. See Table 2 for raw data and number of measurements

Table 2  Raw data of isolated nucleoli Young's moduli of AMDtreated and MG132-treated cells

YM (Pa) Nucleoli number Force curves number

Control

AMD

MG132

30,962 194

26,016 107

36,775 79

1,473

891

645

into isolated nucleoli. The 70-kDa molecules did not penetrate homogenously but exhibited some accumulation in the heterochromatin shell surrounding the nucleoli (Fig. 2). This result was quite surprising because proteins of at least this molecular weight can shuttle between the nucleolus and nucleoplasm by simple diffusion in HeLa, U2OS and CHO cells (Chen and Huang 2001; Tsai and McKay 2005). AFM surface topography imaging revealed that this heterochromatin shell is composed of beads of 270 nm diameters, themselves made of smaller beads of 60 nm diameters (Fig. 3). These observations are concordant with a study by Cheutin et al. (Cheutin et al. 2003), which showed similar structures of 30–50 nm and 250–300 nm, by electron tomography. Those structures, cords (250–300 nm) made of small fibrils (30–50 nm) observed by SEM, are

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described as potential higher-order chromatin organization created by the peripheral nucleolar protein piK67. The electron tomography data of this study clearly show the same beaded structure that we observed with AFM. Furthermore, these levels of chromatin folding have been previously described by several studies (Belmont et al. 1999; Sadoni et al. 2001; Yoshimura et al. 2003). Therefore, the heterochromatin surrounding the nucleolus is probably a tight beaded structure, which could act as a barrier (Fig. 10). Our results are different from those obtained in Xenopus laevis oocytes in which dextran of higher molecular weight penetrates the nucleolus (Handwerger et al. 2005). The most striking difference between nucleoli from Xenopus laevis oocytes and those of HeLa cells is that the former are devoid of a heterochromatin shell, which can explain the difference with our results. Moreover, Handwerger et al. injected dextran molecules into the nucleus, therefore benefiting from the nuclear environment. Our experiments were performed on isolated nucleoli, therefore in the absence of nucleoplasm and the surrounding nuclear chromatin. It is conceivable that the nucleoplasm and/or chromatin motion could create some force on the heterochromatin shell and the nucleolus, therefore facilitating the penetration of larger molecules.

Histochem Cell Biol Fig. 6  Morphological structure of nucleoli in control cells and after RPS6, RPL11, GAPDH and NegC knockdowns. The DFC is labeled through fibrillarin immunostaining (red). The GC is labeled via the GFP–B23 expression (green). Scale bars 5 μm

The nucleolar structure is flexible, and it can be modulated We first showed that it was possible to measure nucleolar stiffness and that it ranges in the magnitude of tens of kPa (Fig. 4). We reasoned that if the nucleolus’ flexibility would be important for the diffusion of molecules in and out of

this body, then it should be possible to modulate it. Our first attempt at this was using inhibitors known to dramatically alter the nucleolar structure. The proteasome inhibitor MG132 creates aggresomes in the nucleolus, which resulted in a higher YM, i.e., stiffer nucleoli (Fig. 5). Conversely, treatment with the transcription inhibitor AMD leads to the

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Histochem Cell Biol

Fig. 7  Ribosomal protein knockdown. a Western Blot revealing the protein amount of RPS6, RPL11, GAPDH and ß-actin in untreated cells (control) and after RPS6, RPL11, GAPDH and NegC knockdowns. b–d Quantifications of the protein amount of RPS6, RPL11

and GAPDH, respectively, after knockdown. Control: blue bar and RPS6, RPL11, GAPDH and NegC knockdowns: red, green, purple and light blue bars, respectively

segregation of the nucleolar compartments: the FC and the DFC have a peripheral localization, whereas the GC remains the central part of the nucleolus (Shav-Tal et al. 2005). Nucleoli treated with AMD had a lower YM, i.e., were softer. The tip of the cantilever was targeted to the middle of the nucleolus. Therefore, in this particular case, the measurement is mainly reflecting the stiffness of the GC itself, the least dense compartment of the nucleolus (Handwerger et al. 2005), which explains the lower YM value (Figs. 5, 10). We then tested whether the nucleolus stiffness could change upon modification of its main function, ribosomal biogenesis, while minimizing its structural alteration. After knockdown of ribosomal proteins RPS6 and RPL11, the calculated YM was higher, i.e., stiffer nucleoli (Fig. 8). Ribosomal protein depletion causes the accumulation of specific pre-rRNA (Robledo et al. 2008). The depletion of RPS6 leads to a processing default of the 5′ external transcribed spacer (ETS) (Fumagalli et al. 2009; Robledo et al.

2008). The depletion of RPL11 leads to a default in the internal transcribed spacer 2 (ITS2) processing (Robledo et al. 2008). The 5′ETS and ITS2 processing occur inside the nucleolus (Carron et al. 2011), so their processing inhibition leads to an accumulation of unprocessed rRNA within the nucleolus. This can explain why the nucleolus is stiffer after ribosomal protein depletion (Fig. 10), although we cannot exclude that the slight morphological changes may have an influence on the measured YM. Is LaminB1 a key player in nucleolar flexibility? After treatment with the rRNA processing inhibitor DRB, the GC is fragmented in what could be assimilated to building blocks (Haaf and Ward 1996; Louvet et al. 2006, 2005). It is then possible to imagine that a skeleton or linkers could maintain the GC building blocks together. LaminB1 has been suggested to be a protein that ensures the plasticity of the nucleolus (Martin et al. 2009). By measuring the stiffness of nucleoli after LaminB1 knockdown, we

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could establish that its stiffness is higher than that of control nucleoli (Fig. 9). Therefore, our study confirms that LaminB1 is an important factor for the nucleolus’ flexibility. LaminB1 is known to tether chromatin to the nuclear envelope, and it interacts with B23 (Martin et al. 2009). It is tempting to speculate that LaminB1 could tether GC building blocks, through interaction with B23, with the chromatin present inside and at the periphery of the nucleolus. This tethering should not be seen as static linkers, but rather as flexible linkers that, when removed, reduce the flexibility of the nucleolus and give rise to a stiffer nucleolus (Fig. 10). Nucleolar stiffness: where does it stand? Our measurements showed that when a force of 2 nN was applied, the nucleolar stiffness ranges from 30 to 60 kPa. With similar loading forces (1–5 nN), Young’s modulus of live cells ranges from 1 to 200 kPa (for review, see Kuznetsova et al. 2007; Radmacher 1997). The nucleolar stiffness is in the range of that of myofibrils, cardiocytes and erythrocytes (40–45 kPa, 5–200 kPa and 14–110 kPa, respectively). When compared to subcellular structures, the nucleolus stiffness is in the same range as that of bundles of actin

Fig. 8  Nucleolar stiffness increases upon ribosomal protein knockdown. Histogram representing the normalized averaged calculated YM of three independent experiments of nucleoli isolated from untreated cells (control, blue bar; 100 %) and after RPS6, RPL11, GAPDH and NegC knockdowns (red, green, purple and light blue bars, respectively; 130, 115, 98 and 103 %, respectively). Experiments were performed in couples: control versus siRNA. See Table 3 for raw data and number of measurements

Table 3  Raw data of isolated nucleoli Young's moduli after ribosomal proteins knockdowns

YM (Pa) Normalized YM  % Nucleoli number Force curves number

filaments and microtubules, ~40 kPa [measured in platelets (Radmacher 1996)]. The nuclear stiffness has mostly been obtained from whole-cell measurements that were performed over the nucleus. The nuclear stiffness, in this situation, ranges from 1 to 10 kPa (Radmacher 1996; Yokokawa et al. 2008, and for review Kuznetsova et al. 2007). The stiffness of isolated nuclei from chondrocytes was measured to be in the magnitude of 1 kPa, which is in the same range as isolated eukaryote chromosomes (isolated from newt lung cells) that have a stiffness between 1 and 5 kPa (Guilak et al. 2000; Houchmandzadeh et al. 1997). If one compares the volume and content of the nucleus to those of the nucleolus, it is obvious that the nucleolus is much more packed. It is, then, not surprising that the nucleolar stiffness is higher than that of the nucleus. This is supported by the work of Handwerger et al. that shows that nucleoli are twice as dense as the nucleoplasm, and by the work of Speil and Kubitscheck, in which they conclude that the lack of penetration of free molecules in the nucleolus, in comparison with the nucleus, is due to the lack of available space (Handwerger et al. 2005; Speil and Kubitscheck 2009). “The nucleolus is formed by the act of building a ribosome” (Mélèse and Xue 1995). One recent study illustrates very well the dynamics of rRNA production by showing that the first three or four steps of rRNA processing occur within a few minutes (Popov et al. 2013). Moreover, the fluid property of the nucleolus has recently been shown (Brangwynne et al. 2011). One can then propose a model where the steady-state nucleolus flexibility is due to a functional ribosome biogenesis (Fig. 10). The stiffness of the nucleolus can change upon different perturbations. After AMD treatment, the nucleoli are softer. When the ribosome biogenesis process is impaired by ribosomal protein knockdowns, the accumulation of unprocessed rRNA leads to stiffer nucleoli. Finally, LaminB1 is also important for the nucleolar stiffness and could play the role of a flexible linker.

Materials and methods Plasmid, cell culture, drug treatments and protein knockdown by siRNA Complementary DNA for B23 was amplified by PCR from HeLa cDNA pool, which was reverse transcribed by

Control

SiRPS6

Control

SiRPL11

Control

SiGAPDH

Control

SiNegC

24,704 100 72

32,024 130 80

43,823 100 63

50,533 115 55

69,409 100 77

67,938 98 83

36,020 100 83

37,048 103 56

557

653

490

391

505

651

688

459

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Fig. 9  Nucleolar stiffness increases upon LaminB1 knockdown. a Labeling of LaminB1 (LB1) (red) on control cells and after 48 h knockdown on a GFP–B23 (green) cell line. Scale bars 10 μm. b After LaminB1 knockdown, nucleoli exhibited a stretched shape, revealed by comparing the shape of the GC (GFP–B23, green) of a control cell to that of a knockdown cell. The DFC is revealed by the detection of fibrillarin (red). Scale bars 5 μm. c Histogram representing the averaged calculated YM of nucleoli isolated from untreated cells (control, blue bar) and after LaminB1 knockdown (red bar). The average calculated YM of the control nucleolus was ~50 kPa, while after LaminB1 knockdown, the calculated YM was higher, ~64 kPa. See Table 4 for raw data and number of measurements

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Histochem Cell Biol

Histochem Cell Biol Table 4  Raw data of isolated nucleoli Young's moduli after LaminB1 knockdown

YM (Pa) Nucleoli number Force curves number

Control

SiLaminB1

51,913 71

64,357 62

548

478

10 % fetal bovine serum (Hyclone). Cells were treated when necessary either by AMD for 2 h at a final concentration of 40 ng/ml or by MG132 for 16 h at a final concentration of 10  μM. SiRNAs targeting RPS6 (ID #45962) and RPL11 (ID #9352) were purchased from Ambion. Silencer negative control #1 (NegC) (Ambion) and Silencer GAPDH (Ambion) were used as negative controls. SiRNA targeting LaminB1 (#HSS106098) was purchased from Invitrogen. Cells were transfected by Lipofectamin2000 (Invitrogen) following the manufacturer’s instructions and collected after 48 h. Antibodies, immunofluorescence and Western blotting

Fig. 10  Model of the relationship between nucleolar structure/function and stiffness. The nucleolus is represented with its three compartments: FC (yellow ellipse), DFC (green ellipse) and GC (pink ellipse). The dashed pink lines represent the internal nucleolar chromatin, and the pink beads at the nucleolar periphery represent the heterochromatin shell. LaminB1 (LB1) is represented by the red croissant shapes. When the ribosome biogenesis is active, the ribosomal subunits (blue shapes of various sizes to represent different stages of maturation) are produced giving some fluidity to the nucleolus and by consequence its steady-state stiffness. When the ribosome biogenesis is inhibited by AMD, the three-nucleolar compartments are segregated. The GC becomes devoid of the FC and DFC that are relocated at its periphery giving access to a softer material. After ribosomal protein knockdown, the flow of ribosomal subunit production is disrupted and accumulation of immature ribosomal subunits occurs, accounting for a stiffer nucleolus. After LaminB1 knockdown, the nucleolus is devoid of its flexible LaminB1 linkers that ensure the junction between the GC and the chromatin accounting for a loss in flexibility

SuperScript II reverse transcriptase (Invitrogen) from the total RNA extracted by RNeasy RNA extraction kit (Qiagen). The cDNA was cloned into pEGFP-C1 vector (Clontech) with KpnI-BamHI sites. The construct for GFP-fused UBF was a kind gift from Dr. M. Okuwaki at Tsukuba University. Transfection into HeLa S3 cells was performed by Effectene transfection reagent (Qiagen) according to the manufacturer’s instructions. The stably expressing cell lines were raised by a single-clone isolation method after screening the transfected cells with G418 (Nacalai). Non-transformed and stably transformed HeLa cells lines (GFP–B23 and GFP– UBF) were grown without antibiotic in complete Dulbecco’s modified Eagle’s medium (Sigma) supplemented with

Antibodies directed a mouse monoclonal to nucleolar proteins were the following: rabbit polyclonal to UBF (Santa Cruz), a mouse monoclonal antibody to fibrillarin (Cytoskeleton and Abcam), mouse monoclonal to nucleolin (Kumeta et al. 2013), rabbit polyclonal to B23 (Abcam), mouse monoclonal to PML (Santa Cruz), rabbit polyclonal to LaminB1 (Abcam), goat polyclonal to GAPDH (Imgenex), rabbit polyclonal to RPS6 (Bethyl), goat polyclonal to RPL11 (Santa Cruz) and mouse monoclonal to ß-actin (Sigma). The secondary antibodies for immunofluorescence were conjugated with FITC or Rhodamine or A568 or Texas red, were from Cappel Laboratories and Molecular Probes. Cells grown on glass coverslips or isolated nucleoli seeded on 0.01 % polyl-lysin (Sigma)-coated coverslips were fixed in 2 % paraformaldehyde for 15 min at room temperature (RT) and permeabilized with 0.5 % Triton X-100 for 5 min. Blocking was performed for 30 min with 0.5 % milk in PBS at RT. The first antibodies were incubated for 45 min at RT and revealed with the fluorochrome-conjugated secondary antibodies. The samples were mounted with Vectashield (Vector Laboratories) containing 20 μM Hoechst 33342 (Nacalai Tesque) for DNA visualization. Optical sections were examined on a confocal laser-scanning microscope (LSM5 PASCAL, Carl Zeiss) with an X63 Plan-Apo objective lens N.A. 1.4. In order to calculate the aspect ratio of the nucleoli, we performed the following procedure with the ImageJ software: the pictures of the GFP–B23 were binarized; the “Analyze particles” option was used in order to automatically detect the nucleoli (objects), which were then measured, and the option “Shape descriptors” was selected, which automatically calculates the aspect ratio of the objects. For Western blotting or Coomassie Blue R250 staining, proteins were first separated on a 12 % one-dimensional SDS–PAGE gel. The gel was then stained by Coomassie, or proteins were transferred on PVDF membrane (Biorad); primary antibodies were incubated either for 3 h at RT or overnight at 4 °C, and secondary antibodies (GE Healthcare) coupled to HRP were incubated for 3 h at RT; HRP signal was revealed by chemiluminescence (Chemi Lumi Kit, Nacalai) and revealed with a LAS-3000 mini (Fujifilm).

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Histochem Cell Biol

Nucleolar isolation

Surface topography imaging by AFM

The protocol was adapted from Prof. Lamond and Diaz laboratories (Andersen et al. 2002; Hacot et al. 2010; Scherl et al. 2002). All buffers are supplemented with antiproteases (Roche). Briefly, HeLa cells were incubated in a hypotonic buffer (10 mM Tris–HCl, pH 7.4; 10 mM NaCl; and 3 mM MgCl2) and incubated on ice for 10 min. Cell lysis was performed with a precooled Dounce homogenizer (Wheaton). Nuclei were collected by centrifugation at 1,200g for 5 min and suspended in 0.25 M sucrose-containing 10 mM Tris–HCl, pH 7.4, 10 mM NaCl and 10 mM MgCl2. Nuclei were then purified by centrifugation at 1,200g for 10 min through a 0.88 M sucrose cushion containing 10 mM Tris–HCl, pH 7.4, 10 mM NaCl and 1.5 mM MgCl2. Purified nuclei were suspended in 0.34 M sucrose-containing 10 mM Tris–HCl, pH 7.4, 10 mM NaCl, 1.5 mM MgCl2 and 0.25 % Nonidet P-40 and sonicated. Nucleoli were then purified by centrifugation at 2,000g for 20 min through a 0.88 M sucrose cushion containing 10 mM Tris–HCl, pH 7.4, 10 mM NaCl and 1.5 mM MgCl2. Purified nucleoli were washed in 0.34 M sucrose-containing 10 mM Tris– HCl, pH 7.4, 10 mM NaCl and 1.5 mM MgCl2. Nucleoli were either suspended in 0.25 M sucrose buffer for sedimentation on PLL-coated glass or Mica, or suspended in Laemmli buffer for 12 % SDS–PAGE gel followed by Coomassie Brilliant Blue R250 staining or Western blotting.

Air conditions: Nucleoli were spotted on a mica surface and dried by air nitrogen gas. Imaging in air was performed on a Nanoscope IV (Digital Instruments) using the J-scanner (AS-130) (scan size 125 × 125 μm; Vertical Range 5 μm) and cantilevers OMCL-AC160TS (Olympus). Fields of 5 × 5 μm were imaged at a scan rate of 1 Hertz (scan rates of 1.6 and 2 Hertz generated the same images). Image analysis was performed using the Nanoscope software. Bead size was measured by tracing a plot profile on the AFM images, and the distance from peak to peak reflects the distance from bead center to bead center. Twenty-eight nucleoli were imaged over a period of several days. The same beaded structures were observed, and the measurements of the beads showed size consistency on the same nucleolus and between nucleoli. Liquid conditions: Nucleoli were isolated from a GFP– UBF stably expressing cell line. Nucleoli were fixed with 2.5 % glutaraldehyde to avoid breaking due to the scanning and spotted on Teflon wells glass slides coated with 0.01 % poly-l-lysin (PLL), stained with DAPI (100 ng/ml) and imaged in PBS. Imaging was performed using BIXAM (Olympus), a fast-scanning AFM mounted on an inverted fluorescence microscope IX71 (Olympus). The possibilities to visualize fluorescence and phase contrast enable for the selection of the sample to be imaged and to target the cantilever on the selected sample. The maximum scan size of the AFM scanner is 2 × 1.5 μm with a vertical range of 0.2 μm, therefore only allowing the surface structure of the nucleolus to be imaged partially. Fluorescence pictures were acquired with an X40 LUCPLFLN N.A. 0.6 and a DP72 CCD camera (Olympus). The sample was imaged at room temperature with BL-AC10DS cantilevers (Olympus) at a scanning rate of one frame per 10 s. Imaging of the nucleolus was repeated three times. Image analysis was performed with the AFM software.

Permeability assay After isolation from a cell line stably expressing GFP– B23, nucleoli were suspended in 0.15 M sucrose buffer containing 100 mM Tris–HCl, pH 7.4, 2 mM MnCl2, 50 mM (NH4)2SO4, 4 mM MgCl2 and antiproteases (Moore and Ringertz 1973). The nucleoli homogenate was split into three aliquots, and neutral dextran (Molecular Probes) of 10, 40 or 70 kDa coupled to tetramethylrhodamine (10 and 40 kDa, #D1816 and #D1842, respectively) or rhodamine (70 kDa, #D1823) was added at a final concentration of 1 μM. Nucleoli were incubated in the presence of Dextran for 3 h at RT on a rotate wheel and protected from light. Nucleoli were then seeded on coated PLL glass coverslips for 30 min at 4 °C, fixed for 15 min at RT with 2 % paraformaldehyde, washed with PBS and mounted in Vectashield containing 20 μM Hoechst 33342 for DNA visualization. This experiment was repeated five times, and 138, 150 and 170 nucleoli were, respectively, analyzed for the 10, 40 and 70 kDa molecules. Optical sections were acquired on a confocal laserscanning microscope (Fluoview FV1200, Olympus) with an X60 Plan-Apo objective lens N.A. 1.42.

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Stiffness measurement by AFM After isolation, nucleoli were seeded on 0.01 % PLLcoated glass bottom dishes (World Precision Instruments) for 30 min on ice in sucrose 0.25 M buffer. For stiffness measurement, the nucleoli or polystyrene beads of 5.25 μm diameter (Spherotech) were incubated in 0.15 M sucrose buffer. The force measurement experiments were performed at RT on a Molecular Force Probe 3D (MFP-3D; Asylum Research) mounted on an inverted optical microscope (IX71; Olympus), allowing simultaneous optical visualization and force measurement. Calibration of the cantilevers was performed in dry condition on a clean glass surface. The spring constant was measured using the thermal method and was between 70 and 90 pN/nm. Then, the

Histochem Cell Biol

sensitivity of the cantilevers was calibrated in liquid: Typically, values comprised between 20 and 40 nm/V. The cantilevers have pyramidal-shaped tips (OMCL-TR400PSA, Olympus), as have been used in several previous studies (Azeloglu and Costa 2011; Kaufmann et al. 2011; Kuznetsova et al. 2007; Radmacher 1996; Solon et al. 2007; Yokokawa et al. 2008). The tip of the pyramid is represented on the top of the cantilever by the intersection of a cross. Force distance measurements were recorded with a trigger force of 2 nN and a velocity of 4 μm/s. Individual nucleoli were selected randomly. Ten force curves were recorded per nucleolus and were registered on 30 control nucleoli and 30 drug-treated or knockdown nucleoli per experiment. To calculate the Young’s modulus, the force curves were then fitted in the SPIP software (Image Metrology), for an indentation of 0–2 nN using the Hertz–SnedE 2 don model FSneddon = π2 (1−ν 2 ) tan (α)(s0 − s) , where E represents Young’s modulus, ν Poisson’s ratio of 0.5, α tip half-cone opening angle 17.5º, s0 point of zero indentation and s0−s indentation. Nucleoli with aberrant force curves and aberrant fits are discarded from the analysis. The YM of one nucleolus is the averaged YM of the pertinent fits obtained from the ten recorded force curves. Then, the YM of the control and drug-treated or knockdown nucleoli is averaged per experiment. Each set of experiments, i.e., drug or knockdown, plus control nucleoli were repeated at least three times, averaged and illustrated as histograms with standard deviations. Cantilevers with a spherical indenter, 1.98-μm diameter polystyrene bead, were also used (see Fig. 4) (CP-PNPL-PS, NanoAndMore GmbH). The spring constant was between 30 and 50 pN/ nm. The sensitivity of the cantilevers was between 60 and 80 nm/V. Force distance measurements were recorded with a trigger force of 2 nN and a velocity of 4 μm/s. To calculate the Young’s modulus, the force curves were fitted in the SPIP software (Image Metrology), for an indentation of 0–2 nN using the sphere indentation Hertz model 3 4 Esurface  FHertz = 3 (1−ν 2 ) Rtip (s0 − s) 2, where E denotes Young’s modulus for the surface, ν Poisson’s ratio of 0.5, Rtip ball radius of the tip, s0 point of zero indentation and s0−s indentation. The analysis was then performed as described for the pyramidal indenter. Acknowledgments E. L. was supported by a postdoctoral fellowship for foreign researchers and a Grant-in-Aid for Scientific Research from the Japanese Society for the Promotion of Sciences (JSPS). M. K. was supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Spying minority in biological phenomena” (#24115512) from MEXT Japan. K. T. was supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Molecular basis of host cell competency in virus infection” (#24115003) from MEXT Japan. We are thankful to Olympus Co., Japan, for technical help and advice with the BIXAM imaging; to Kei Murata and Yaron Silberberg for technical help and advice; and to Nicolas Joannin for critical reading of the manuscript.

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