Nuclear Actin Puncta in Senescent Cells

: Both cellular senescence and the nuclear actin cytoskeleton participate in a plethora of physiological and pathological activities. However, their connection remains elusive. Recently, it is becoming increasingly apparent that filamentous actin plays important functions within the nucleus. Particularly, micron-scaled actin filaments (hereinafter referred to as F-actin) in the nucleus are of the essence in DNA damage repair, cell fate determination and age-related diseases etc. Latrunculin B (latB), a pharmaceutically applicable macrolide, can induce nuclear F-actin assembly. Now it is unclear how the nuclear actin skeleton responds to latB stimulation in senescent cells. Here, by time-lapse images, Doxo-induced senescent IMR-90 cells showed a large number of nuclear actin puncta, then the nuclear actin puncta become brighter and larger in size upon latB treatment. Some actin puncta even served as the elongation bases for the nuclear F-actin. Therefore, our results link the nuclear actin skeleton to cell aging and reveal the effect of latB treatment on the nuclear actin skeleton in aging cells.


Introduction
Tremendous researches have shown that human health and biological systems change with aging [1][2][3].Many diseases such as cardiovascular disease, cancer, diabetes, osteoarthritis and Parkinson's disease, are closely connected with organismal aging [4][5][6][7].Therefore, for better medication and drug discovery, it is important to understand the mechanism of aging and find out the effects of aging at the cellular level.
Recently, researches have been focusing on dissecting the roles of micron-scaled nuclear filamentous actin under different physiological or stress conditions [9][10][11][12][13][14].Although F-actin is widely studied in the cytosol in most cells, the presence of nuclear F-actin, only recently, has been associated with diverse nuclear functions [10,13,[15][16][17][18]. Transient (usually appears and exists in the order of minutes, disassembles afterwards) assembly of nuclear F-actin is found during serum stimulation, cell spreading, mitotic exit, DNA damage repair and T cell activation [15][16][17][18][19][20].In contrary, persistent nuclear F-actin has been proved to correlate with some diseases/stress statuses (such as human skeletal muscle disease intranuclear rod myopathy, Parkinson's disease and Huntington's disease) [21].The existence of persistent nuclear F-actin inhibits global transcription by RNA Polymerase II and reduces cell proliferation [22].Therefore, it is interesting and important to unravel the relationship between cellular senescence and the nuclear actin cytoskeleton.
However, the relationship between nuclear actin dynamics and cellular senescence and its role in latB trratment in senescent cells are unknown.In this study, we aimed to solve this problem.

Establishment of a Cellular Senescence Model
To study cellular senescence, we first established a doxorubicin-induced cellular senescence model [8].Cellular senescence was confirmed by an increase staining of senescence-associated beta-galactosidase (SA-β-gal) and decrease staining of the cell proliferation marker Ki-67 (Figure 1 A and 1B) [8,29].

RNA Sequencing Comparing Young and Senescent Cells
Next, to understand the potential changes during cellular senescence, we performed RNA sequencing experiments comparing the transcriptomes of the early passage young cells and the doxorubicin-induced senescent cells.Curiously, the KEGG enrichment analysis showed the enrichment of actin cytoskeleton-related genes among other pathways, suggesting changes of actin regulation or the actin cytoskeleton during cellular senescence (Figure 2).It is noteworthy that, without latB treatment, a relatively high percentage of dot-like actin structures (hereinafter referred to as actin puncta) was observed in senescent cells rather than in young cells, suggesting a sub-population of actin structures exist during cellular senescence (Figure 3A, lower panel and Figure 3B).Enlargement of the senescent cell nucleus showed that some actin puncta displayed hollow shapes (Figure 3A, second panel enlarged image, blue arrows), sizes of which were close to the microscopy diffraction limit.Confocal time-lapse microscopy imaging showed that nuclear actin puncta were relatively stable without latB treatment (Figure 3B, yellow arrows).After latB treatment, many actin puncta in the senescent cells became brighter and larger in size (Figure 3C, blue arrows).Some actin puncta served as the elongation bases for the nuclear F-actin (Figure 3C, yellow arrows).

Discussion
Dissecting the fundamental mechanisms and effects of cellular senescence is beneficial for the basic aging researches and the development of anti-aging pharmaceutics.The relationship between cellular senescence and the nuclear Factin cytoskeleton in mammalian cells is unclear.Most previous researches focused on the cytoplasmic F-actin rather than the nuclear actin counterpart.Although a number of works suggested a link between the cytoplasmic F-actin and cellular senescence [30,31], there were limited pieces of evidence suggesting a direct relationship between nuclear Factin and cellular senescence [8,32].In this study, we have addressed a new link between cellular senescence and micron-scaled nuclear F-actin assembly under latB treatment (Figure 4).By using doxorubicin-induced senescent human diploid fibroblast IMR-90 as a cellular senescence model, we found an enrichment of the transcriptional change of the actin cytoskeleton regulation genes during cellular senescence.By time-lapse images, we also found nuclear actin puncta that participate in nuclear actin reorganization upon latB treatment in the senescent cells.Our results showed that some actin puncta displayed hollow shapes.Superresolution microscopy will be needed to further decipher the structure and function of these nuclear actin puncta.Collectively, this study opens a new perspective to understand the nuclear actin changes during cellular senescence and demonstrates a new link between cellular senescence, nuclear actin cytoskeleton and latB application.

RNA Sequencing and Data Analysis
Total RNAs of young and doxorubicin-induced senescent IMR-90 cells were isolated for RNA sequencing experiments and analysis.cDNA library construction and sequencing were performed by Shenzhen B.G.I. company using the BGISEQ-2000 platform.The expression levels for each gene were normalized to fragments per kilobase of exon model per million mapped reads (FPKM) using RNA-seq by Expectation Maximization (RSEM).Data analysis was done by using the online Dr. Tom system.

Sample Preparation and Immunofluorescence Staining
IMR-90 cells were seeded in the glass-based 35 mm confocal dishes (NEST).After cells reached ~70% confluence, cells were fixed for 10 min by 4% paraformaldehyde (freshly prepared).Fixation solution was decanted and IMR-90 cells were washed three times in PBS.IMR-90 cells were permeabilized for around 5 min by 0.1% Triton X-100 solution.Permeabilization solution was decanted and IMR-90 cells were washed three times in PBS.Then IMR-90 cells were blocked for around 1 h by 5% BSA at room temperature and further incubated with primary antibody for 2 h at room temperature.Solution was decanted and IMR-90 cells were washed three times (5 min each wash) in PBS.IMR-90 cells were Incubated with the secondary antibody goat anti-rabbit IgG Alexa Fluor-488 (Invitrogen, A-11034) in 1% BSA for 1 h at room temperature in dark.Solution was decanted and cells were washed three times (5 min each wash) in PBS.Nuclear DNA was labeled by DAPI.The following primary antibodies were used in Figure 1B: Ki-67, abcam (ab15580).

Confocal Microscopy Imaging
IMR-90 cells were seeded in the glass-based 35 mm confocal dishes (NEST) at appropriate cell density overnight.After changing media with Latrunculin B (Cayman, 1 µM), cells were maintained in a 37°C chamber with 5% CO2 during the entire imaging process.Confocal microscopy systems were utilized for capturing fluorescent images.

Figure 2 .Figure 3 .
Figure 2. KEGG Enrichment of RNA sequencing results shows a change in some actin regulation genes.RNA sequencing was done comparing the young and senescent IMR-90 cells 2.3.Nuclear Actin Puncta in Senescent IMR-90 Cells

Figure 4 .
Figure 4. Schematic of distinct nuclear F-actin response upon latB treatment