1 Nuclear bundle / cable containing actin during yeast meiosis

Actin polymerizes to form filaments/cables for motility, transport, and structural framework in a cell. Recent studies show that actin polymers are present not only in cytoplasm, but also in nuclei of vertebrate cells, and their formation is induced in response to stress. Here, by electron microscopic observation with rapid freezing and high-pressure freezing, we found a unique bundled structure containing actin in nuclei of budding yeast cells undergoing meiosis. The nuclear bundle/cable during meiosis consists of multiple filaments with a rectangular lattice arrangement often showing “feather-like” appearance. The bundle is immuno-labeled with anti-actin antibody and sensitive to an actin-depolymerizing drug. Like cytoplasmic bundles, nuclear bundles with actin are rarely seen in pre-meiotic cells and spores, and are induced during meiotic prophase-I. The formation of the nuclear bundles/cables is independent of meiotic DNA double-stranded breaks. We speculate that nuclear bundles/cables containing actin play a role in nuclear events during meiotic prophase I.


Introduction
In the cytoplasm, as a cytoskeletal protein, actin polymerizes to form a filament (F-actin) for various cellular functions such as motility, division, phagocytosis, endocytosis, and membrane trafficking 1 . Dynamics of cytoplasmic actin filaments are highly regulated by various factors in different environments. Actin is also present in nuclei 2 . Actin monomer functions as a component of several chromatin-remodeling complexes for transcription and other nuclear events 3-6 . cases, nuclear bundles span across through whole nucleus (~1 µm, see also Fig. 6-section 5). The longer bundles consist of multiple short bundles, rather than a single linear bundle (see Fig. 6-section 5).
We note a recent preprint by Gan's group which, by using cryo-electron tomography, showed a nuclear bundle in meiotic yeast cells, which referred to as "meiotic triple helix" (Ma et al. BioRxiv, 10.1101/746982v1). The nuclear bundle found here is structurally similar to the meiotic triple helix.

Spatial arrangement of nuclear bundles/cables in meiosis
To get more spatial information on nuclear bundles, we checked serial sections of a nucleus in yeast cells at 4 h in meiosis ( Fig. 5 and 6). To achieve in-depth freezing in specimens, we froze cells under a high pressure 35 . With highpressure freezing specimens suitable for sectioning were obtained. Importantly, we did not see any change of sub-cellular structures including cables in yeast cells prepared by either rapid freezing or high-pressure freezing (compare Fig. 2 with Fig. 5 and 6). We often detected multiple nuclear bundles in different sections of 60 nm (Fig. 5, 6), indicating that the cables are an abundant nuclear structure with three-dimensional arrangement of cables in a nucleus (Fig. 5). In some sections, a long bundle that spans entire nucleus is observed (Fig. 6-sections 5 and 6), and the end of the cable is likely to attach to the NE (arrows in Fig. 6section 4-6).
Nuclear bundles often accommodate branched-like filaments or bundles, which look like "feather" (Fig. 2g, h). At this resolution some branched filaments look attached a lateral side of filaments in a main bundle while the other filaments are not attached. We measured an angle between main bundles and branched bundles/filaments (Fig. 4d, Supplementary Fig. 1). At 4 h, angles between the main bundle and branch-like filaments are 25°-40° with sub-peak of 15°-20° ( Fig.   4d; 38°±19° [n=31], median=34°). We noticed that branch-like filaments from a single bundle are oriented same direction, suggesting the presence of the directionality of the cable.

Nuclear bundles are similar to cytoplasmic bundles in meiosis
We also detected cables/bundles in cytoplasm of meiotic yeast cells (Fig. 2g, i).
Structurally, the bundles in cytoplasm are similar to those in nuclei (compare Fig.   2g, h). The diameter of a single thin filament in cytoplasmic bundles is around 7-8 nm (Supplementary Fig. 2a; 7.1±1.0 nm [n=21]), which is similar to that of nuclear bundles (Fig. 4a). The length of cytoplasmic bundles in sections (mean=304±179 nm [n=121], median=238 nm; Fig. 4c) is not different from that in cytoplasm (P=0.64, Mann-Whitney's U-test). These indicate that nuclear and cytoplasmic bundles/cables are structurally indistinguishable. While nuclear bundles form complicated 3D structure, cytoplasmic bundles form less such the structure (Fig. 3b).

Nuclear bundles by chemical fixation
Yeast cells are surrounded with thick cell walls, which impair penetration of staining reagents such as osmic tetroxide. We also stained spheroplasts of meiotic yeast cells with osmic acid after fixation with glutaraldehyde (without any freezing, Supplementary Fig. 3) and, in some cases, prepared serial sections for EM observation (Supplementary Fig. 4). With this procedure, the mitochondria are contrasted highly ( Supplementary Fig. 3). Membrane structure such as nuclear membrane was partially deformed, possibly due to hypo-osmotic conditions. Importantly, even under this condition, we could detect the cables/bundles in both the nucleus and the cytoplasm of cells during meiotic prophase-I, whose structures and arrangement are similar to those obtained by the freeze-substitution method.

Nuclear and cytoplasmic bundles are formed during prophase-I
We checked the presence of nuclear bundles in different stages of meiosis (Fig.   4e). As a control, we measured nuclei containing microtubules. The nuclear microtubules are seen at 0 h and sections positive to microtubules are increased slightly during meiotic prophase-I. Nuclear bundles appear earlier during meiosis than cytoplasmic bundles (Fig. 4e). At 0 h before the induction of meiosis in which most of cells are G1, we detected few bundles in nuclei ( These indicate that the formation of nuclear bundles as well as cytoplasmic bundles are independent of meiotic DSBs induced by Spo11, thus meiotic recombination and the SC.

Previous EM analyses of mitotic cells of both budding and fission yeasts have
shown three distinct actin sub-cellular structures; rings, cables, and patches as well as less-defined filasome 21-24 . As a less-defined actin-related structure, filasome is a cytoplasmic structure of less electron-dense areas with a vesicle in the center ( Supplementary Fig. 6), a novel actin-containing membrane-less subcellular structure in cytoplasm originally found in fission yeast 24 .
We initially found that EM images of meiotic nuclear and cytoplasmic Previously, immuno-EM confirmed the presence of actin in the bundles in cytoplasm of yeasts 38 . We performed immuno-gold labeling of chemical fixation sections using anti-actin antibody, which provides less clear image compared to conventional EM (Fig. 8c, d). Although not extensive, we found clustered gold labels on the bundles in nuclei and in cytoplasm (Fig. 8c, d). In a nucleus, in addition to the bundles, we often detected the particles on densestained area containing filament-like structure, which might be bundles.
Importantly, nuclear bundles of freeze fixation specimens showed more gold particles than other nuclear area ( Supplementary Fig. 7). These suggest that both nuclear and cytoplasmic bundles contain actin. We also detected gold particles on less electron-dense areas in cytoplasm without ribosomes, which might correspond to the filasome (Fig. 8c).

Discussion
In this study, by using TEM with rapid freezing-fixation, we found nuclear bundles/cables containing actin in budding yeast cells undergoing the physiological program of meiosis. We could also detect the bundles in cytoplasm, which are also induced during prophase I. Since we used rapid freezing to preserve structures inside of cells, it is unlikely that the cable is an artifact produced by specimen preparation, which might be induced by external stress and/or staining. Moreover, we also detected the bundles in nuclei fixed with chemicals without freezing (Supplementary Fig. 3 and 4). We also noted that cryo-electron tomography showed a nuclear bundle in meiotic yeast nuclei, which is similar to the nuclear bundle described in this paper (Ma et al. BioRxiv, 10.1101/746982v1).
Both nuclear and cytoplasmic bundles are structurally similar, consisting of multiple parallel filaments (Fig. 9a). Nuclear (and cytoplasmic) bundles seem to contain a unique arrangement of filaments, in which alternate pattern of 1 and 2 filaments is observed in a cross section of the cables (Fig. 9b). This alternate pattern provides rectangular/square arrangement of filaments in a single bundle.
The distance of inter-filaments is mainly 10-15 nm. Although we sometimes observed branch-like structure of the bundle, we have not had any solid evidence on the branching of the filaments at current resolution of EM.
Nuclear bundles elongate up to 1 µm ( Fig. 4c and Fig. 6). This long bundle consists of several bundles in a linear array, rather than a single bundle, suggesting a self-assembly property of the bundle. We speculate that nuclear bundles are assembled into a long cable with lateral attachment (or branching).
Several bundles are present in a single nucleus of meiotic cells (Fig. 5 and 6), indicating that nuclear bundles are abundant with three-dimensional arrangement during late prophase I. The nuclear bundles formed during meiosis appear to have a unique ultra-structure: multiple bundles/cables accommodate threedimensional arrangement, possibly through lateral interaction among the bundles and/or branch-like configuration. If abundant, how these cables are packed in the context of nucleoplasm with meiotic chromosome structures such as SCs remains to be determined. Unfortunately, we could not efficiently detect SCs in our cryo-sections, which could be detected in silver-staining of fixed meiotic cells 30-32 .
Nuclear bundles are induced from very early meiotic prophase-I such as 2-h post induction of meiosis and are present to at least by meiosis I nuclear division. The bundles are abundant not only in nuclei, but also in cytoplasm particularly during late prophase-I (Fig. 3b). On the other hand, we rarely see nuclear or cytoplasmic bundles in a G1 diploid cell (pre-meiotic) or in spores (Fig.   2a, i). During meiosis, the formation of nuclear bundles starts at 2 h post-induction of meiosis, prior to DSB formation, which begins at ~3 h, indicating that nuclear bundle formation is not be associated with DSB formation during yeast meiosis. What is a role of nuclear bundles? Actin polymerization in cytoplasm is involved in chromosome motion during prophase-I in budding yeast 42 .
Cytoplasmic actin cables promote meiotic chromosome motion through SUN-KASH protein ensembles in the NE 29 , which is sensitive to the LatB 27 . Our results here suggest that nuclear bundles also play a role in the movement of meiotic chromosomes. Alternatively, nuclear bundles may protect nuclear structures from external forces generated during meiosis as seen in Xenopus oocyte 16 , by providing a rigid structure that resists mechanical stress generated through chromosome motions. We speculate that nuclear bundles may form threedimensional structure through self-assembly inside of meiotic nuclei. Further studies are necessary to reveal nature and function of nuclear bundles containing actin in meiotic cells.

Rapid Freezing for transmission electron microscopy
Specimens for freeze-substitution electron microscopy were prepared according to previously described method 45, 46 , with slight modifications. Cells were harvested by centrifugation. The cell pellets were sandwiched between two copper disks (3 mm in diameter). Specimens were quickly frozen with liquid propane using a rapid freezing device (KF80; Leica, Vienna, Austria). Specimens were freeze-substituted in cold absolute acetone containing 2% osmium tetroxide (OsO4) at -80°C for 48-72 h and were then warmed gradually (at -40°C for 4 h, at -20°C for 2 h, at 4°C for 2 h and at room temperature for 2 h) and washed with absolute acetone and rinsed with QY-2. Substitution for embedding was infiltrated with Quetol-812 mixture (10, 30, 50, 70, 80 and 90 and 100% pure resin). The specimens polymerized at 60°C for 2 days.

High pressure freezing fixation for transmission electron microscopy
The specimens for high-pressure freeze-substitution electron microscopy were prepared according to previously described method 47 with slight modifications.
The pelleted cells were pipetted into aluminum specimen carriers (Leica) and frozen in a high-pressure freezing machine HPM-010 (BAL-TEC, Liechtenstein).
The cells were transferred to 2% OsO4 in cold absolute acetone. Substitution fixation was carried out at −90°C for over 80 h. After the fixation, the specimens were warmed gradually (at -40°C for 2 h, at -20°C for 2 h, at 4°C for 2 h and at room temperature for 1 h) and washed with absolute acetone and then with exchange to 0.1% uranyl acetate in absolute acetone. After the staining, specimens were washed with absolute acetone and rinsed with QY-2.
Substitution and embedding were described in above.

Image processing and measurement
Measurement of diameter of actin cables and distance between actin cables was carried out using ImageJ Fiji. 51 . Cropped regions of TEM images were converted into binary images and noises were removed. Filaments more than 10nm 2 in size and 0.5 -1.0 in circularity were recognized with analyze particles function. Minor axis lengths of each particle were regarded as diameters of filaments. Distances between filaments were calculated using the central coordinate of particles.

Statistics
Statistical significance for Length of actin cables between that in nucleus and cytoplasm was analyzed using the Mann-Whitney's U-test. The null hypothesis was that there exists no variation of the length between nucleus and cytoplasm.

Statistical significance for existence of actin cables in cells with and without
LatB treatment was analyzed using Fisher's exact test. The null hypothesis was that LatB treatment did not affect existence of actin cables in cells. Two-sided Pvalue was shown.

Data availability
Strains are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article and figures.
EM images are available upon request.