Branching and intercellular communication in the Section V cyanobacterium Mastigocladus laminosus, a complex multicellular prokaryote

1 This article is protected by copyright. All rights reserved. Branching and intercellular communication in the Section V cyanobacterium 1 Mastigocladus laminosus, a complex multicellular prokaryote1 2 Dennis J Nürnberg1, Vicente Mariscal2, Jamie Parker1, Giulia Mastroianni1, Enrique Flores2, and 3 Conrad W Mullineaux1# 4 1School of Biological and Chemical Sciences, Queen Mary University of London, Mile End 5 Road, London E1 4NS, United Kingdom 6 2Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas 7 and Universidad de Sevilla, Américo Vespucio 49, E-41092 Seville, Spain 8 # For correspondence. E-mail [email protected]; Tel. (+44) 20 7882 3645; Fax (+44) 20 9 8983 0973. 10 Running title: Intercellular communication in Mastigocladus laminosus 11 Keywords: cyanobacteria, true branching, Mastigocladus laminosus, intercellular 12 communication, SepJ, cell differentiation13 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/mmi.12506 Accepted Article 2 This article is protected by copyright. All rights reserved. Summary 14 The filamentous Section V cyanobacterium Mastigocladus laminosus is one of the most 15 morphologically complex prokaryotes. It exhibits cellular division in multiple planes, resulting in 16 the formation of true branches, and cell differentiation into heterocysts, hormogonia and necridia. 17 Here, we investigate branch formation and intercellular communication in M. laminosus. 18 Monitoring of membrane rearrangement suggests that branch formation results from a 19 randomised direction of cell growth. Transmission electron microscopy reveals cell junction 20 structures likely to be involved in intercellular communication. We identify a sepJ gene, coding 21 for a potential key protein in intercellular communication, and show that SepJ is localised at the 22 septa. To directly investigate intercellular communication, we loaded the fluorescent tracer 5-23 carboxyfluorescein diacetate into the cytoplasm, and quantified its intercellular exchange by 24 Fluorescence Recovery after Photobleaching. Results demonstrate connectivity of the main 25 trichome and branches, enabling molecular exchange throughout the filament network. Necridia 26 formation inhibits further molecular exchange, determining the fate of a branch likely to become 27 a hormogonium. Cells in young, narrow trichomes and hormogonia exhibited faster exchange 28 rates than cells in older, wider trichomes. Signal transduction to coordinate movement of 29 hormogonia might be accelerated by reducing cell volume. 30 Introduction 31 Among non-eukaryotes, cyanobacteria have achieved a high degree of morphological complexity 32 and diversity. It is remarkable that multicellularity in this phylum evolved early in Earth history, 33 possibly as early as the “Great Oxygenation Event” that took place around 2.48 to 2.32 billion 34 years ago and allowed the development of all life we know today (Bekker et al., 2004; Tomitani 35 Accepted Article 3 This article is protected by copyright. All rights reserved. et al., 2006; Konhauser et al., 2011; Schirrmeister et al., 2011). According to their morphology, 36 cyanobacteria have been divided into five sections, including unicellular forms (Section I and II), 37 filamentous (Section III and IV) and filamentous-branching forms (Section V) (Rippka et al., 38 1979). Organisms of Section IV (also known as order Nostocales (Komárek and Anagnostidis, 39 1989)) and V (Stigonematales (Anagnostidis and Komárek, 1990)) show additionally the ability 40 to undergo cell differentiation, forming heterocysts (specialised cells for nitrogen fixation), and 41 sometimes also akinetes (resting cells for survival of adverse environmental conditions), and 42 hormogonia (motile filaments for dispersal and symbiosis competence). Accordingly, 43 multicellularity in cyanobacteria has been defined by three processes: cell-cell adhesion, 44 intercellular communication and terminal cell differentiation (Flores and Herrero, 2010). 45 While our understanding of multicellularity in Section IV has deepened considerably by studying 46 Anabaena sp. PCC 7120 as model organism (for review see (Flores and Herrero, 2010)), there is 47 little known about cyanobacteria of Section V. The best understood organism within this section 48 is probably Mastigocladus laminosus, which is a major component of epilithic microbial mats at 49 White Creek, Yellowstone National Park, USA (Miller et al., 2006), and can be found in 50 geothermal sites and hot springs worldwide with an upper temperature limit of 63°C (Cohn, 51 1862; Schwabe, 1960; Castenholz, 1976; Melick et al., 1991; Finsinger et al., 2008; Soe et al., 52 2011; Mackenzie et al., 2013). 53 Under most conditions, M. laminosus forms a dense network of intertwined narrow and wide 54 trichomes (also known as type II (secondary) and type I (primary) trichomes respectively 55 (Schwabe, 1960)). While cells of narrow trichomes have a uniform cylindrical shape, cells of 56 wide trichomes are rounded and pleomorphic, giving usually rise to true branches, the 57 characteristic feature for cyanobacteria of Section V (Anagnostidis and Komárek, 1990; Golubić 58 Accepted Article 4 This article is protected by copyright. All rights reserved. et al., 1996; Komárek et al., 2003). Microfossil records from Rhynie, Aberdeenshire, Scotland 59 support the presence of this complex morphotype already around 400 million years ago (Croft 60 and George, 1959). True branching includes several different types, which have been named for 61 simplicity after their morphological appearance, including “T”, “V”, “X”, and (reverse) “Y” 62 branching (Anagnostidis and Komárek, 1990; Golubić et al., 1996). Lateral “T”, “V” and 63 (reverse) “Y” branches are comprised of cylindrical cells (Desikachary, 1959; Fogg et al., 1973; 64 Golubić et al., 1996). Branches can differentiate into motile hormogonia which are released from 65 the main filament by death and disintegration of the branching point (Balkwill et al., 1984). The 66 released hormogonia glide away from the parental colony, and finally form new colonies by 67 differentiating into spherical cells, which give rise to new lateral branches (Hernandez-Muniz and 68 Stevens, 1987; Robinson et al., 2007). According to ultrastructural investigations, cell division in 69 M. laminosus and Fischerella ambigua differs from that seen in filamentous cyanobacteria of 70 Sections III and IV (Thurston and Ingram, 1971; Martin and Wyatt, 1974; Nierzwicki et al., 71 1982). Rounded cells in wide trichomes were suggested to be separated by their surrounding 72 sheath (Martin and Wyatt, 1974). This would suggest that their filamentous character is only 73 maintained by sheath material, so cyanobacteria of Section V may not represent the pinnacle of 74 development among cyanobacteria but rather a primitive and basic form linking coccoid and 75 filamentous forms (Martin and Wyatt, 1974). Under nitrogen deprivation almost every cell can 76 differentiate into a heterocyst, following no regular spacing pattern, often forming multiple 77 contiguous heterocysts in wide trichomes (MCH; sets of heterocysts, connected to each other 78 without vegetative cells in between) (Nierzwicki-Bauer et al., 1984a; Nierzwicki-Bauer et al., 79 1984b; Stevens et al., 1985). This raises the question of how cells communicate in M. laminosus. 80 Here we investigate intercellular communication in M. laminosus by loading the fluorescent 81 tracer 5-carboxyfluorescein diacetate (5-CFDA) into the cytoplasm, and performing Fluorescence 82 Accepted Article 5 This article is protected by copyright. All rights reserved. Recovery after Photobleaching (FRAP) experiments to observe intercellular exchange of dye 83 molecules, using methodology previously applied to filamentous cyanobacteria of Sections III 84 and IV (Mullineaux et al., 2008). In the Section IV cyanobacterium Anabaena sp. PCC7120, 85 there is rapid diffusion of dye molecules between the cytoplasms of neighbouring cells, 86 dependent on the septum-localised proteins SepJ (FraG), FraC and FraD (Mullineaux et al., 2008; 87 Merino-Puerto et al., 2011). Here, we address the questions of whether the branch and the main 88 trichome communicate in M. laminosus, and whether exchange depends on the cell morphotype. 89 Our results demonstrate that branch and main trichome exchange molecules, and that exchange 90 depends on the cell morphology, showing that M. laminosus makes a particularly complex 91 network of intercellular communication. 92 Just recently, the genome sequences of several species of Section V were published (Dagan et al., 93 2013; Shih et al., 2013).It has been suggested that no signature proteins specific to any of the 94 complex morphologies exist (Shih et al., 2013), and that branching might be mainly a result of 95 expressing a very few proteins which affect the regulation of cell division genes and/or the 96 localisation of their proteins (Dagan et al., 2013). Here we follow the localisation of the 97 cytoplasmic membrane during the process of branch formation to gain further insights into this 98 complex event. 99 100 Accepted Article 6 This article is protected by copyright. All rights reserved. Results and Discussion 101 Randomisation of the axis of growth results in different types of branching 102 M. laminosus forms two morphologically distinct types of true branches: (reverse) “Y” (Fig. 1A) 103 and “T” branches (Fig. 1B). Both types of branches are not only present in the same culture, but 104 even in the same filament (not shown), raising the question of whether the differences between 105 the two types are merely superficial, or whether they arise from different developmental 106 processes. 107 We approached this question by visualising the cytoplasmic membrane during branch formation 108 using confocal microscopy and transmission electron microscopy. For confocal microscopy we 109 stained cells with the fluorescent dye FM1-43FX, which highlights the cytoplasmic membrane in 110 cyanobacteria (Schneider et al., 2007). Our results indicate that the (reverse) “Y” and “T” 111 branches are topologically equivalent: in both cases branch formation is initiated by the growth of 112 a cell in a direction other than the main axis of the filament (Fig. 1A,C). This is generally 113 followed by septum formation across the mid-line of the cell, as is the usual rule in bacteria (Fig. 114 1A). The result of septum formation is that one of the daughter cells is connected to three cells: 115 two in the main trichome and one in the developing branch (Fig. 1A,B). A “T” branch results 116 from cell elongation in a direction roughly perpendicular to the filament axis (Fig. 1B), whereas a 117 (reverse) “Y” branch results from cell elongation at a more acute angle to the filament axis (Fig. 118 1A,C), but the two cases are only superficially different. This implies that branching is the result 119 of a randomisation of the direction of cell elongation. When cell elongation is constrained to 120 occur along the filament axis, branch formation is repressed. A developmental switch leading to 121 randomisation of the direction of cell elongation allows branches to form. Our conclusion is 122 consistent with the message from recent genome sequence analyses of several cyanobacteria of 123 Accepted Article 7 This article is protected by copyright. All rights reserved. Section V, which did not detect any specific Section V signature proteins (Dagan et al., 2013; 124 Shih et al., 2013). Furthermore, it was shown by Singh and Tiwari (1969) that true branching can 125 be induced in the non-branching filamentous cyanobacterium Nostoc linckia (Roth) Born. et Flah. 126 (Section IV) by random mutagenesis using ultraviolet irradiation, which implies that branching 127 can be induced by loss of gene function rather than being the result of a complex developmental 128 programme. This fits with our conclusion that branching results from the selective relaxation of a 129 stringent control over the direction of cell elongation. We could detect no obvious patterns in the 130 spacing of branches, which appears to be random. Terminal cells of wide trichomes do not branch 131 and have a different, elongated shape (Fig. 1D), which has been described as a terminal hair 132 (Anagnostidis and Komárek, 1990). The attachment of another cell, or even a cell fragment, at 133 the terminus of the filament is enough to inhibit cell elongation (Fig. S1). A similar topology can 134 be found e.g. in the filamentous cyanobacterium Oscillatoria acuminate (Section III; Geitler, 135 1960). 136 137 M. laminosus forms a complex cellular network of communication mediated by septosomes 138 Until now it has remained unknown whether the branch and the main trichome communicate in 139 cyanobacteria of Section V. A prerequisite for answering this question is to load a hydrophilic 140 fluorescent molecule into the cytoplasm of cells of both the branch and the main trichome. For 141 Anabaena sp. PCC 7120 two fluorescent molecules that can be used as tracers have been 142 described in detail, calcein (Mullineaux et al., 2008) and 5-carboxyfluorescein diacetate (5-143 CFDA; Mariscal et al., 2011). In both cases, the tracer is added to the cell culture in an esterified 144 form which is non-fluorescent and hydrophobic enough to be cell-permeant. Hydrolysis of the 145 ester groups by cytoplasmic esterases converts the molecule to a fluorescent form which is 146 Accepted Article 8 This article is protected by copyright. All rights reserved. trapped in the cytoplasm because it is too hydrophilic to traverse lipid bilayers. Hence it can be 147 used to probe intercellular exchange of hydrophilic molecules via protein channels at the cell 148 junctions (Mullineaux et al., 2008; Mariscal et al., 2011). We tested both fluorescent tracers for 149 M. laminosus. The efficiency of cell labelling with 5-CFDA was much higher than that with 150 calcein (data not shown), and hence suitable for further studies. 151 The question of connectivity between branch and main trichome cannot be answered simply by 152 photobleaching and following fluorescence recovery of the cell at the branch point, as 153 fluorescence recovery might be possible from three directions. We found that branch formation 154 could be induced by agitating the cell culture for 24h in fresh medium, consistent with a previous 155 observation that branch formation is induced by conditions favouring rapid growth (Thurston and 156 Ingram, 1971). Cells were then loaded with 5-CFDA, and fluorescence of entire short branches 157 bleached by scanning the region of the branch at increased laser intensity (Fig. 2). All the cells in 158 a branch, regardless whether they formed a “T” (Fig. 2) or (reverse) “Y” branch (not shown), 159 showed recovery. A quantified analysis of recovery, shown for a specimen “T” branch in Fig. 2, 160 in which the bleached out branch was defined as one region of interest (ROI), reveals that 161 recovery is mediated by cells from both sides next to the branching point. The fluorescence 162 intensity decreases in the adjacent cells over time. Accordingly our results demonstrate that 163 trichomes of M. laminosus form a complex interconnected cell communication network. A newly 164 formed branch remains connected to its main trichome. 165 The exchange of molecules between the cytoplasm of cells requires the presence of cell-to-cell 166 connecting structures penetrating the peptidoglycan layer and the plasma membranes of both 167 cells (e.g. Merino-Puerto et al., 2011; Lehner et al., 2013). The occurrence of such structures, 168 which have been termed septosomes (or microplasmodesmata), has been well characterized in 169 Accepted Article 9 This article is protected by copyright. All rights reserved. Anabaena spp. by various methods, such as thin-section TEM (e.g. Wildon and Mercer, 1963), 170 freeze-fracture EM (Giddings and Staehelin, 1978; Giddings and Staehelin, 1981) and electron 171 tomography (Wilk et al., 2011), but it has remained unclear whether septosomes exist in M. 172 laminosus. A first indication of their presence in M. laminosus was given by Marcenko (1962), 173 who could identify pores with an average diameter of 15 nm in the cross-walls of isolated cell 174 wall sacculi. Our electron micrographs of thin sections through the septal regions of M. 175 laminosus clearly show structures pervading the septa between vegetative cells (Fig. 3A) and 176 between heterocysts and vegetative cells (Fig. 3B). Different methods of sample preparation for 177 TEM can also be used to reveal insights into the composition of intercellular channels (Wilk et 178 al., 2011). Our results are in good agreement with the proposed proteinaceous nature of the 179 septosomes found in Anabaena sp. PCC 7120 (Wilk et al., 2011). While septosomes appear as 180 positively stained structures in a KMnO4-based preparation method (Fig. 3A), they are negatively 181 stained in an OsO4-based preparation (Fig. 3B). 182 Although the presence of pores penetrating the septum was shown earlier in Stigonema 183 hormoides, Fischerella muscicola, and F. ambigua, which belong to cyanobacteria of Section V, 184 it has been suggested that these pores do not pierce the underlying plasma membranes, and 185 accordingly do not mediate a direct connection of the protoplasts (Thurston and Ingram, 1971; 186 Butler and Allsopp, 1972). Our results from fluorescent dye exchange, however, show the 187 connectivity of the cytoplasm throughout the entire filament network, which is likely achieved by 188 structures resembling septosomes. 189 190 Accepted Article 16 This article is protected by copyright. All rights reserved. increase, they can be distinguished from other filament types by their distinctly smaller cell size 329 (Campbell and Meeks, 1989). Accordingly, we defined hormogonia of M. laminosus by cell 330 diameter, cell length, and the diameter to length ratio, resulting in a group of cells characterised 331 by an average cell diameter of 2.76 ± 0.31 µm, an average cell length of 3.61 ± 0.88 µm, and a 332 diameter to length ratio of 0.80 ± 0.19 (n=80). While the mean exchange coefficient E is similar 333 to that found between cells in narrow trichomes (E = 0.159 ± 0.072 s-1) and meets the expectation 334 that cellular communication is fast in hormogonia, the flux coefficient F is significantly lower, 335 showing a value similar to that found in wide trichomes (F = 3.60 ± 2.47 µm3 s-1) (Table 1). 336 Therefore communication between cells in hormogonia is rapid in the sense that intercellular 337 diffusion of molecules is fast enough to lead to rapid changes in the cytoplasmic concentration of 338 putative signalling molecules. However, as compared to cells in the parental filament, this rapid 339 communication is achieved by reducing the cell volume rather than by accelerating the flux of 340 molecules across the cell junctions. Therefore signal transduction to coordinate movement (and 341 possibly other aspects of the biology) of hormogonia is probably accelerated by the reduction of 342 the cell volume rather than by increased flux of signalling molecules across the cell junctions. 343 Barriers to cell-cell communication are formed early in the life cycle of M. laminosus 344 The release of a hormogonium from its parental trichome is mediated by the formation of 345 releasing, dead cells, called necridia, which are possibly best studied in Oscillatoria/Microcoleus 346 spp. (Kohl, 1903; Lamont, 1969; Brown et al., 2010). Beside heterocysts (Meeks et al., 2002), 347 necridia are one of the few known developmental “dead ends” among prokaryotes, and can be 348 seen as a basic form of programmed cell death (apoptosis). Since a necridium can be found early 349 after the branching event in the growing narrow trichome (Fig. 5A-C), we investigated whether 350 exchange of molecules is still possible between the branch and the main trichome. After loading 351 Accepted Article 17 This article is protected by copyright. All rights reserved. the fluorescent tracer 5-CFDA into the cytoplasm, we bleached out the fluorescence of the part of 352 the branch which was separated by the necridium and followed its change in fluorescence 353 intensity (Fig. 5A). We did not observe any recovery of the bleached region within 24 s (Fig. 5A) 354 indicating that a necridium inhibits cell-cell communication completely. Accordingly, the fate of 355 a trichome is determined early after branch formation; molecular exchange not being possible 356 once a necridium is formed. 357 To get an impression of the complexity of this event and its importance for the filamentous 358 network, we investigated the position of necridium formation. Necridia can be easily detected by 359 enhanced red fluorescence in the region of formation, possibly as a result of degradation of the 360 photosynthetic apparatus (Figs. 5B-D and 6A). Necridia also show brighter staining with the 361 membrane dye FM1-43FX (Fig 5B-D), probably because leakiness of the cytoplasmic membrane 362 allows the dye to penetrate to the interior of the cell. We observed that necridia are not only 363 located in the branching point, as reported in an earlier study (Balkwill et al., 1984), but also in 364 various other positions within narrow trichomes, such as the beginning of the recently formed 365 branch (Fig. 5C), indicating that almost any cell within a filament can differentiate into a 366 necridium. Even within the same filament several necridia can be found, contiguous or separated 367 only by a single cell (Fig. 5D), suggesting that necridia formation in M. laminosus seems to 368 follow no regular pattern of spacing and distribution. 369 370 The final release of the trichome is a complex mechanism of remodelling the septal region on 371 both sides of the necridium. To avoid cell death of the entire cellular network by efflux of 372 molecules, the membranes of the neighbouring cells have to stay intact, and the channels/pores 373 have to be closed. Electron micrographs confirm that the plasma membrane and outer membrane 374 Accepted Article 18 This article is protected by copyright. All rights reserved. are sealed at the terminal cell (Fig. 6B). Although we observed that necridia usually consist only 375 of a single cell, they can be formed by two cells (Fig. 5B, D). A possible mechanism to prevent 376 efflux of molecules in other cyanobacteria, e.g. Symploca muscorum and M. vaginatus might be 377 by the synthesis of an additional cell wall layer at the new terminus (“calyptra”; Pankratz and 378 Bowen, 1963; Lamont, 1969). It remains yet to be investigated whether this structure also exists 379 in M. laminosus. 380 Filament breakage, however, is only possible by the disintegration of the membranes of the 381 necridium. To investigate this process we used the cytoplasmic membrane stain FM1-43FX (Fig. 382 5B,C,D) and the DNA stain Hoechst 33258 (Fig. 6A). The fluorescence micrographs indicate that 383 during necridium formation the cytoplasmic membrane of the necridium deteriorates mainly from 384 one terminus of the cell (Fig. 5B,C,D), leaving an “open” and empty (DNA-free) cell attached to 385 the released filament (Fig. 6), while only a small part of membranes remains at the terminus of 386 the parental filament. 387 Concluding remarks 388 Our results demonstrate that M. laminosus represents a complex cellular network, in which the 389 main trichome and branches communicate via intercellular connections which resemble 390 septosomes. We observed that exchange between cells within a culture is regulated, depending on 391 cell morphology. Young, narrow trichomes exhibited rapid exchange rates among cells, while 392 old, wide trichomes showed reduced rates. Accordingly, wide trichomes might not only provide a 393 platform for the outgrowth of branches, but they might also support the growth of branches by 394 supplying metabolites in the presence and absence of a combined nitrogen source. Under nitrogen 395 deprivation heterocysts can be found frequently in the branching region, sometimes even in the 396 branching start, forming a heterocyst with three cyanophycin plugs. The integrity of the filament 397 Accepted Article 19 This article is protected by copyright. All rights reserved. network is only interrupted by the formation of necridia, which inhibit further molecular 398 exchange, and hence determine the fate of a developing branch likely to become a hormogonium, 399 soon after the branching event. Interestingly, signal transduction to coordinate movement of the 400 released hormogonia might be accelerated by the reduction in cell volume. 401 Cell differentiation seems to be generally less regulated than in Section IV cyanobacteria; the 402 formation of heterocysts (Nierzwicki-Bauer et al., 1984a; Nierzwicki-Bauer et al., 1984b; 403 Stevens et al., 1985) and necridia (this study) seem to follow no regular spacing and distribution 404 pattern. Our analyses of the different types of branches also suggest a degree of randomness in 405 cell development. We hypothesise that “T” and “Y” branches are basically equivalent: the 406 different forms simply result from loose control of the positioning of the cell elongation 407 machinery. 408 As a possible component of the cell-cell connecting structures we identified SepJ. The protein 409 consists of the typical three domain architecture found in other filamentous, heterocyst-forming 410 cyanobacteria (Section IV), and immunofluorescence labelling revealed its localisation at the 411 septa. A cell in the branching point exhibits three SepJ spots, suggesting that although M. 412 laminosus shows branching, the septa are similar to those described in Anabaena sp. PCC 7120. 413 Earlier studies had suggested that rounded cells in wide trichomes are completely separated by 414 their surrounding sheath (Thurston and Ingram, 1971; Martin and Wyatt, 1974; Nierzwicki et al., 415 1982) which would imply a lack of communication between these cells. According to our 416 ultrastructural and FRAP analyses this is however not the case. M. laminosus shows intercellular 417 communication and highly-structured cell junctions between cells of various shapes, forming a 418 complex network of cell communication. The hypothesis that cyanobacteria of Section V 419 represent a primitive and basic form linking coccoid and filamentous forms (Martin and Wyatt, 420 Accepted Article 20 This article is protected by copyright. All rights reserved. 1974), is not supported by our study. Our results from cell division and intercellular 421 communication experiments indicate that Section V cyanobacteria are similar to cyanobacteria of 422 Section IV. Section IV and V cyanobacteria show the highest degree of morphological 423 complexity and diversity within the phylum. 424 425 Accepted Article 21 This article is protected by copyright. All rights reserved. Experimental Procedures 426 Organism, medium, and growth. 427 Mastigocladus laminosus SAG 4.84 was originally isolated by G. H. Schwabe from a thermal 428 spring of Reyhjanes/Isafjord, Iceland in 1967. The cyanobacterium was grown in liquid 429 Castenholz D medium (Castenholz, 1988) at 40°C, under constant white light at approximately 430 20 µE m-2 s-1. To provide constant agitation cultures were bubbled with sterile air or shaken (100 431 rpm). For studies of intercellular communication between branch and main trichome, a resting 432 culture was used to inoculate Castenholz D medium. After 24 h of growth cultures were loaded 433 with 5-CFDA, and its transfer demonstrated by FRAP. Heterocyst differentiation was induced by 434 growth for up to 96 h in Castenholz ND medium (Castenholz, 1988), which lacks combined 435 nitrogen, under the above described conditions. 436 Labelling with fluorescent dyes. 437 For molecular exchange experiments, cells were labelled with the fluorescent tracers 5-438 carboxyfluorescein diacetate (5-CFDA; Molecular Probes) or calcein (Molecular Probes). 1 ml of 439 M. laminosus culture was harvested by centrifugation (3000xg, 2 min.), washed twice and 440 resuspended in 1 ml fresh growth medium, and mixed with 12 µl of a 1 mg ml-1 5-CFDA or 441 calcein, acetoxymethyl ester solution in dimethylsulphoxide. The suspension with 5-CFDA was 442 incubated for 30 min at 40°C in the dark with shaking in an orbital incubator at 80 rpm, and the 443 suspension with calcein for 90 min under the same conditions. To remove the fluorescent dyes, 444 the cells were washed three times in growth medium, and incubated for another 30 min in 1 ml of 445 medium at 40°C in the dark under gentle shaking (80 rpm). After a final washing step, cells were 446 spotted onto a Castenholz D 1.5 % (w/v) agar plate, and excess solution was removed. To 447 Accepted Article 22 This article is protected by copyright. All rights reserved. maintain the growth conditions throughout the experiment media and plates were preheated to 448 40°C. 449 Two additional fluorescent dyes, FM1-43FX and Hoechst 33258, were used to visualise specific 450 cellular components. FM1-43FX stains the outer and cytoplasmic membranes of cyanobacteria 451 (Schneider et al., 2007), while Hoechst 33258 interacts with DNA. For labelling, we washed cells 452 once in fresh growth medium, and added 1 µl Hoechst 33258 (1 mg ml-1; Bisbenzimide H33258; 453 AppliChem) and/or 2.5 µl FM1-43FX (0.1 mg ml-1; Molecular Probes) to 0.5 ml of culture. The 454 suspension was incubated for 10 min at room temperature and washed twice with growth medium 455 prior mounting on 1.5 % (w/v) agar with Castenholz D or ND medium. Surplus medium was 456 removed. 457 Confocal microscopy and Fluorescence Recovery after Photobleaching (FRAP). 458 For confocal microscopy, small blocks of agar were placed in a custom-built temperature-459 controlled sample holder covered with a glass cover slip. Cells were visualised with the Leica 460 laser-scanning confocal microscope SP5 using a x63 oil-immersion objective (Leica HCX PL 461 APO lambda blue 63.0x1.40 OIL UV), and an excitation wave length of 488 nm for 5-CFDA, 462 calcein and FM1-43FX, and 355 nm for the Hoechst 33258-DNA complex. Chlorophyll a 463 fluorescence (autofluorescence) and dye fluorescence were imaged simultaneously, using 464 different emission detection ranges (455-495 nm for Hoechst 33258, 500-527 nm for 5-CFDA 465 and calcein, 570-595 nm for FM1-43X, and 670-720 nm for the autofluorescence). For imaging, 466 we used a 95 µm confocal pinhole, giving a resolution of 0.8 µm in the Z-direction (full width at 467 half-maximum of the point-spread function), whereas we opened the pinhole maximal for FRAP 468 measurements (600 µm, an optical section thickness of 4.2 µm respectively). After taking an 469 Accepted Article 23 This article is protected by copyright. All rights reserved. initial image (pre-bleach), the region of interest (ROI) was bleached by increasing the laser 470 intensity and zooming into the ROI, and recovery was recorded. Images were taken typically in 471 0.534 s intervals. FRAP data were analysed as described in (Mullineaux et al., 2008) to estimate 472 the exchange coefficient E, using Image Pro Plus 6.3 (Media Cyberentics Inc.) and SigmaPlot 473 10.0 (Systat Software Inc.). 474 Electron Microscopy. 475 Cultures were harvested by centrifugation (3000xg; 2 min) and fixed for 2 h at room temperature 476 with 4% (w/v) glutaraldehyde in 100 mM phosphate buffer pH 7.3. To remove the fixative, cells 477 were washed three times with 100 mM phosphate buffer. After embedding in 2% (w/v) low-478 gelling temperature agarose, samples were cut in one- to two-millimetre cubic blocks, and post-479 fixed with 2% (w/v) potassium permanganate dissolved in distilled water over night at 4°C. 480 Samples were washed with distilled water until the supernatant remained clear, and dehydrated 481 through a graded ethanol series (1x15 min 30%, 1x15 min 50%, 1x15 min 70%, 1x15 min 90% 482 and 3x20 min 100%). Two washes for 5min with propylene oxide were performed prior 483 infiltration with Araldite for 1 h and with fresh Araldite over night. Polymerisation was achieved 484 by incubation at 60-65°C for 48 h. Alternatively, cells were fixed for 2h at room temperature in 485 100 mM phosphate buffer pH 7.0 containing 3% (w/v) glutaraldehyde, 1% (w/v) formaldehyde 486 and 0.5% (w/v) tannic acid, washed with phosphate buffer, and incubated in 2% (w/v) OsO4 in 487 phosphate buffer over night. Dehydration was performed using a graded acetone series as 488 described for ethanol prior to embedding in Araldite. Thin sections were cut with a glass knife at 489 a Reichert Ultracut E microtome and collected on uncoated, 300 mesh copper grids. High 490 contrast was obtained by poststaining with saturated aqueous uranyl acetate and lead citrate 491 Accepted Article 24 This article is protected by copyright. All rights reserved. (Reynolds, 1963) for 4 min each. The grids were examined in a JOEL JEM-1230 transmission 492 electron microscope at an accelerating potential of 80kV. 493 494 DNA isolation, gene amplification, and sequencing. 495 Genomic DNA from M. laminosus was extracted using the protocol of Morin et al. (2010) with a 496 cell homogenization step prior cell lysis. The cells were harvested by centrifugation (3,000xg, 5 497 min) and resuspended in fresh growth medium. Homogenization of the culture was achieved by 498 multiple passages through a 0.8 mm needle with the help of a syringe. The cells were collected by 499 centrifugation (3000xg, 5 min), and genomic DNA was isolated as previously described (Morin 500 et al., 2010) 501 To identify sepJ in M. laminosus oligonucleotide primers were designed as follows: Since there 502 were no sepJ sequences known from M. laminosus and other species of Section V, we assumed a 503 similarity in its sequence and localisation to other filamentous, heterocyst-forming cyanobacteria. 504 sepJ nucleotide sequences from Anabaena sp. PCC 7120, Anabaena variabilis ATCC 29413, 505 'Nostoc azollae' 0708 and Nostoc punctiforme PCC 73102 were obtained from the NCBI 506 GenBank and aligned with Clustal W 2.1 (Larkin et al., 2007). The first 20 nucleotides built 507 primer fw_mlam_sepJ (5’-atggggcgatttgagaagcg-3’) (Fig. S2B). The reverse primer was designed 508 on the assumption that hetR is located downstream of sepJ. Available partial hetR sequences from 509 cyanobacteria of Section V (Fischerella spp. and Chlorogloeopsis spp.) were used for a Clustal 510 W 2.1 alignment, and resulted in primer rev_mlam_hetR (5’-gttgcggctgcatctaaaaa-3’) (Fig. S2A). 511 The optimal annealing temperature for sepJ amplification was determined by using a gradient 512 PCR with the Promega PCR Master Mix. PCR products were resolved by electrophoresis in a 1% 513 Accepted Article 25 This article is protected by copyright. All rights reserved. (w/v) agarose gel. The final amplification was performed with the Roche Expand High Fidelity 514 PCR System using the following conditions for a 50µl reaction: 94°C for 2 min, 15x cycle 1 515 (94°C for 45 sec; 48.9°C for 45 sec; 72°C for 4 min), 15x cycle 2 (94°C for 45 sec; 59°C for 45 516 sec; 72°C for 4 min) and 1x cycle 3 (94°C for 45 sec; 57°C for 45 sec; 72°C for 10 min). 517 The amplified product was checked on a 1% (w/v) agarose gel, and purified with the QIAquick 518 PCR purification kit (QIAGEN) for sequencing. 519 Nucleotide sequence accession number. 520 The identified sepJ sequence has been deposited in the GenBank database under the accession 521 number KF729033. 522 Sequence analysis. 523 The sepJ DNA sequence was translated into the corresponding amino acid sequence using the 524 ExPASy translate tool (http://web.expasy.org/translate/). A BlastP search was performed in order 525 to identify orthologous sequences (Altschul et al., 1997). Further sequence analyses were 526 performed using the TMHMM server 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) in order to 527 predict the localisation of transmembrane helices, TRUST (Szklarczyk and Heringa, 2004) for 528 the detection of internal repeats, and Coils/PCoils (http://toolkit.tuebingen.mpg.de/pcoils) for the 529 identification of potential coiled-coiled regions in SepJ. 530 Immunofluorescence labelling and sample examination. 531 Cells were harvested by centrifugation (3000xg; 2 min) and resuspended in fresh growth medium. 532 Cultures were transferred onto 0.2 µm Nucleopore membranes, which were subsequently placed 533 onto poly-L-lysine coated slides. For fixation the slides were incubated in 50-ml plastic Falcon 534 Accepted Article 32 This article is protected by copyright. All rights reserved. Smith, D.J., Timonen, H.J., Jaffe, D. a, Griffin, D.W., Birmele, M.N., Perry, K.D., et al. (2013) 696 Intercontinental dispersal of bacteria and archaea by transpacific winds. Appl Env Microbiol 79: 1134–697 1139. 698 Soe, K.M., Yokoyama, A., Yokoyama, J., and Hara, Y. (2011) Morphological and genetic diversity of the 699 thermophilic cyanobacterium, Mastigocladus laminosus (Stigonematales, Cyanobacteria ) from Japan and 700 Myanmar. Phycol Res 59: 135–142. 701 Stevens, S.E.J., Nierzwicki-Bauer, S.A., and Balkwill, D.L. (1985) Effect of nitrogen starvation on the 702 morphology and ultrastructure of the cyanobacterium Mastigocladus laminosus. J Bacteriol 161: 1215–8. 703 Szklarczyk, R., and Heringa, J. (2004) Tracking repeats using significance and transitivity. Bioinformatics 704 20: 311–317. 705 Thurston, E.L., and Ingram, L.O. (1971) Morphology and fine structure of Fischerella ambigua. J Phycol 706 7: 203–210. 707 Tomitani, A., Knoll, A.H., Cavanaugh, C.M., and Ohno, T. (2006) The evolutionary diversification of 708 cyanobacteria: molecular-phylogenetic and paleontological perspectives. Proc Natl Acad Sci USA 103: 709 5442–5447. 710 Wildon, D.C., and Mercer, F. V (1963) The ultrastructure of the vegetative cell of blue-green algae. Aust J 711 Biol Sci 16: 585–596. 712 Wilk, L., Strauss, M., Rudolf, M., Nicolaisen, K., Flores, E., Kühlbrandt, W., and Schleiff, E. (2011) 713 Outer membrane continuity and septosome formation between vegetative cells in the filaments of 714 Anabaena sp . PCC 7120. Cell Microbiol 49: 1–12. 715 Zhang, J.-Y., Chen, W.-L., and Zhang, C.-C. (2009) hetR and patS , two genes necessary for heterocyst 716 pattern formation , are widespread in filamentous nonheterocyst-forming cyanobacteria. Microbiology 717 155: 1418–1426. 718 719 Accepted Article 33 This article is protected by copyright. All rights reserved. 720 Table 1. Exchange (E) and flux coefficients (F) for 5-CFDA in M. laminosus. measurement mean E [s-1] (± s.d.) mean F [µm3s-1] (± s.d.) 1. cells in narrow trichomes 0.159 ± 0.072 7.65 ± 5.19 2. cells in wide trichomes 0.058 ± 0.044 3.60 ± 2.47 3. cells in hormogonia 0.132 ± 0.063 3.14 ± 1.04 t-tests indicate that E and F are significantly different in (1) and (2) (P<0.00001). Number of experiments performed for (1) 27, (2) 57, and (3) 9 Accepted Article 34 This article is protected by copyright. All rights reserved. Figure Legends FIG.1. Different types of branching in M. laminosus, and their development revealed by confocal (A,B,D) and transmission electron microscopy (C). Two main types of branching are present in M. laminosus, namely (reverse) “Y”- (A) and “T”-branching (B). While the division plane is localised parallel to the main filament axis in “T”-branching (B), it remains nearly transversal to the main filament axis during the formation of a (reverse) “Y”-branch (A,C). Cells at the terminus of the main trichome grow into the direction of the main filament but alter their cell shape (D). A,B. The images show FM1-43 FX fluorescence (yellow; left), chlorophyll a fluorescence (magenta, middle), and an overlay of both (right). Scale bars, 5µm. C. Electron micrograph of a thin section prepared with KMnO4; scale bar, 2µm. D. Bright-field image; scale bar, 5µm. FIG. 2. Intercellular transfer of 5-CFDA between branch and main trichome in a “T”-branch of M. laminosus. A. FRAP image sequence. Only 5-CFDA fluorescence is shown. The left image was recorded prior to bleaching (pre). After bleaching out fluorescence in the branch (t = 0), the change in fluorescence intensity was followed over 32 s. Scale bars, 5 µm. B. Quantitation of cell fluorescence of the FRAP sequence displayed in A. Regions of interest (ROI) were defined as shown in the left image, the “T”-branch comprised of three cells was considered as one ROI (“cell 3”). The corresponding fluorescence recovery is indicated in the right graph. Scale bar, 5 µm. FIG. 3. Electron micrographs of ultra thin sections through the septal region of M. laminosus, and localisation of SepJ by immunofluorescent labelling. A,B. Electron micrographs indicate the presence of structures connecting the cytoplasm of adjacent cells (septosomes; arrows) in M. Accepted Article 35 This article is protected by copyright. All rights reserved. laminosus, using either a KMnO4- (A) or a OsO4-based preparation method (B). Septosomes are present between vegetative cells (A), and between vegetative cells and heterocysts (B). Note that the outer membrane does not enter the septum. CP – cyanophycin plug. Scale bars, 200nm. C. Localisation of SepJ in M. laminosus by immunofluorescent labelling. SepJ is localised in distinct spots in the septa between two adjacent vegetative cells. A branching point shows three distinct regions where SepJ is present (arrow). The images show SepJ immunolabelling (green; left), fluorescence of chlorophyll a (magenta; middle), and an overlay of both (right). Scale bars, 5µm. FIG. 4. Position of heterocysts in filaments of M. laminosus. A-D. Heterocysts (arrows) can be found either in the branching start of “T”- (A) or (reverse) “Y”-branches (C), or in the new formed lateral branches (B and D, respectively). Note the position of cyanophycin granules in the heterocysts. They are always located close to cells they are connected with. Scale bars, 5 µm. E. Double heterocyst in a narrow trichome of M. laminosus (arrows). Cyanophycin granules are located at each pole of the cell, resulting in two cyanophycin plugs between two heterocysts. Scale bar, 10 µm. F. Electron micrograph of an ultra-thin section of a heterocyst in M. laminosus. A cyanophycin plug (CP) is present in the neck region. Rearrangements of thylakoid membranes are visible. The sample was prepared using the KMnO4 method. Scale bar, 1µm. FIG. 5. Function of necridia in intercellular communication, and their localisation in filaments of M. laminosus. A. FRAP image sequence of 5-CFDA loaded cells. Intercellular transfer is inhibited between main trichome and branch by the formation necridium (grey arrow).The left image was recorded prior to bleaching (pre). After bleaching out fluorescence in the branch (t = 0), recovery was followed over 24 s. The ROI is indicated with a white arrow. Scale bars, 5µm.B- D. Position of necridia and reorganisation of membranes in filaments of M. laminosus. Necridia Accepted Article 36 This article is protected by copyright. All rights reserved. (arrows) can be found in the branching start (B), at the beginning of a recently formed branch (C), or at various positions within a narrow trichome (D). Their position follows no regular pattern. Two necridia can be even found in a single filament, separated only by one vegetative cell (D).The images show FM1-43 FX fluorescence (yellow; left), autofluorescence (magenta, middle) and an overlay of both (right). Scale bars, 5 µm. FIG. 6. Appearance of necridia after filament release. A. Localisation of DNA in a hormogonium of M. laminosus. DNA was visualised by staining cells with Hoechst 33258 (blue). Autofluorescence is shown in magenta. An overlay with the bright-field image illustrates the position of DNA in the hormogonium, while a dead part remains at the end of the released filament (arrow). Scale bars, 5µm. B. Electron micrograph of an ultra-thin section of a branch terminus after filament breakage via necridium formation. Note that plasma membrane (PM) and outer membrane (OM) are sealed at the terminal cell to prevent cell death by molecule efflux. The sample was prepared with the method based on KMnO4. Scale bar, 1 µm. 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