Academic literature on the topic 'Chlamydomonas, flagella, ultrastructure analysis'

Intraflagellar transport (IFT), which is the bidirectional movement of particles within flagella, is required for flagellar assembly. IFT particles are composed of ∼16 proteins, which are organized into complexes A and B. We have cloned Chlamydomonas reinhardtii and mouse IFT46, and show that IFT46 is a highly conserved complex B protein in both organisms. A C. reinhardtii insertional mutant null for IFT46 has short, paralyzed flagella lacking dynein arms and with central pair defects. The mutant has greatly reduced levels of most complex B proteins, indicating that IFT46 is necessary for complex B stability. A partial suppressor mutation restores flagellar length to the ift46 mutant. IFT46 is still absent, but levels of the other IFT particle proteins are largely restored, indicating that complex B is stabilized in the suppressed strain. Axonemal ultrastructure is restored, except that the outer arms are still missing, although outer arm subunits are present in the cytoplasm. Thus, IFT46 is specifically required for transporting outer arms into the flagellum.

Mutations in human CEP290 cause cilia-related disorders that range in severity from isolated blindness to perinatal lethality. Here, we describe a Chlamydomonas reinhardtii mutant in which most of the CEP290 gene is deleted. Immunoelectron microscopy indicated that CEP290 is located in the flagellar transition zone in close association with the prominent microtubule–membrane links there. Ultrastructural analysis revealed defects in these microtubule–membrane connectors, resulting in loss of attachment of the flagellar membrane to the transition zone microtubules. Biochemical analysis of isolated flagella revealed that the mutant flagella have abnormal protein content, including abnormal levels of intraflagellar transport proteins and proteins associated with ciliopathies. Experiments with dikaryons showed that CEP290 at the transition zone is dynamic and undergoes rapid turnover. The results indicate that CEP290 is required to form microtubule–membrane linkers that tether the flagellar membrane to the transition zone microtubules, and is essential for controlling flagellar protein composition.

Several studies have indicated that the central pair of microtubules and their associated structures play a significant role in regulating flagellar motility. To begin a molecular analysis of these components we have generated central apparatus-defective mutants in Chlamydomonas reinhardtii using insertional mutagenesis. One paralyzed mutant recovered in our screen, D2, is an allele of a previously identified mutant, pf16. Mutant cells have paralyzed flagella, and the C1 microtubule of the central apparatus is missing in isolated axonemes. We have cloned the wild-type PF16 gene and confirmed its identity by rescuing pf16 mutants upon transformation. The rescued pf16 cells were wild-type in motility and in axonemal ultrastructure. A full-length cDNA clone for PF16 was obtained and sequenced. Database searches using the predicted 566 amino acid sequence of PF16 indicate that the protein contains eight contiguous armadillo repeats. A number of proteins with diverse cellular functions also contain armadillo repeats including pendulin, Rch1, importin, SRP-1, and armadillo. An antibody was raised against a fusion protein expressed from the cloned cDNA. Immunofluorescence labeling of wild-type flagella indicates that the PF16 protein is localized along the length of the flagella while immunogold labeling further localizes the PF16 protein to a single microtubule of the central pair. Based on the localization results and the presence of the armadillo repeats in this protein, we suggest that the PF16 gene product is involved in protein-protein interactions important for C1 central microtubule stability and flagellar motility.

The central pair of microtubules and their associated structures play a significant role in regulating flagellar motility. To begin a molecular analysis of these components, we generated central apparatus-defective mutants in Chlamydomonas reinhardtii using insertional mutagenesis. One paralyzed mutant recovered in our screen contains an allele of a previously identified mutation, pf20. Mutant cells have paralyzed flagella, and the entire central apparatus is missing in isolated axonemes. We have cloned the wild-type PF20 gene and confirmed its identity by rescuing the pf20 mutant phenotype upon transformation. Rescued transformants were wild type in motility and in axonemal ultrastructure. A cDNA clone containing a single, long open reading frame was obtained and sequenced. Database searches using the predicted 606-amino acid sequence of PF20 indicate that the protein contains five contiguous WD repeats. These repeats are found in a number of proteins with diverse cellular functions including beta-transducin and dynein intermediate chains. An antibody was raised against a fusion protein expressed from the cloned cDNA. Immunogold labeling of wild-type axonemes indicates that the PF20 protein is localized along the length of the C2 microtubule on the intermicrotubule bridges connecting the two central microtubules. We suggest that the PF20 gene product is a new member of the family of WD repeat proteins and is required for central microtubule assembly and/or stability and flagellar motility.

Intraflagellar transport (IFT) is the bidirectional movement of multipolypeptide particles between the ciliary membrane and the axonemal microtubules, and is required for the assembly, maintenance, and sensory function of cilia and flagella. In this paper, we present the first high-resolution ultrastructural analysis of trains of flagellar IFT particles, using transmission electron microscopy and electron-tomographic analysis of sections from flat-embedded Chlamydomonas reinhardtii cells. Using wild-type and mutant cells with defects in IFT, we identified two different types of IFT trains: long, narrow trains responsible for anterograde transport; and short, compact trains underlying retrograde IFT. Both types of trains have characteristic repeats and patterns that vary as one sections longitudinally through the trains of particles. The individual IFT particles are highly complex, bridged to each other and to the outer doublet microtubules, and are closely apposed to the inner surface of the flagellar membrane.

Centrin, a 20-kD phosphoprotein with four calcium-binding EF-hands, is present in the centrosome/basal body apparatus of the green alga Chlamydomonas reinhardtii in three distinct locations: the nucleus-basal body connectors, the distal striated fibers, and the flagellar transition regions. In each location, centrin is found in fibrous structures that display calcium-mediated contraction. The mutant vfl2 has structural defects at all of these locations and is defective for basal body localization and/or segregation. We show that the vfl2 mutation is a G-to-A transition in the centrin structural gene which converts a glutamic acid to a lysine at position 101, the first amino acid of the E-helix of the protein's third EF-hand. This proves that centrin is required to construct the nucleus-basal body connectors, the distal striated fibers, and the flagellar transition regions, and it demonstrates the importance of amino acid 101 to normal centrin function. Based on immunofluorescence analysis using anti-centrin antibodies, it appears that vfl2 centrin is capable of binding to the basal body but is incapable of polymerizing into filamentous structures. 19 phenotypic revertants of vfl2 were isolated, and 10 of them, each of which had undergone further mutation at codon 101, were examined in detail. At the DNA level, 1 of the 10 was wild type, and the other 9 were pseudorevertants encoding centrins with the amino acids asparagine, threonine, methionine, or isoleucine at position 101. No ultrastructure defects were apparent in the revertants with asparagine or threonine at position 101, but in those with methionine or isoleucine at position 101, the distal striated fibers were found to be incomplete, indicating that different amino acid substitutions at position 101 can differentially affect the assembly of the three distinct centrin-containing fibrous structures associated with the Chlamydomonas centrosome.

Abstract The length of the flagella of Chlamydomonas reinhardtii cells is tightly regulated; both short-flagella and long-flagella mutants have been described. This report characterizes ten long-flagella mutants, including five newly isolated mutants, to determine the number of different loci conferring this phenotype, and to study interactions of mutants at different loci. The mutants, each of which was recessive in heterozygous diploids with wild type, fall into three unlinked complementation groups. One of these defines a new gene, lf3, which maps near the centromere of linkage group I. The flagellar length distributions in populations of each mutant were broad, with the longest flagella measuring four times the length of the longest flagella seen on wild-type cells. Each of the ten mutants had defective flagellar regrowth after amputation. Some of the mutants showed no regrowth within the time required for wild-type cells to regenerate flagella completely. Other mutants had subpopulations with rapid regeneration kinetics, and subpopulations with no observable regeneration. The mutants were each crossed to wild type to form temporary quadriflagellate, dikaryon cells; in each case the long flagella were rapidly shortened in the presence of the wild-type cytoplasm, demonstrating that the mutants were recessive, and that length control could be exerted on already assembled flagella.

ABSTRACT Cilia and flagella are cell organelles that are highly conserved throughout evolution. For many years, the green biflagellate alga Chlamydomonas reinhardtii has served as a model for examination of the structure and function of its flagella, which are similar to certain mammalian cilia. Proteome analysis revealed the presence of several kinases and protein phosphatases in these organelles. Reversible protein phosphorylation can control ciliary beating, motility, signaling, length, and assembly. Despite the importance of this posttranslational modification, the identities of many ciliary phosphoproteins and knowledge about their in vivo phosphorylation sites are still missing. Here we used immobilized metal affinity chromatography to enrich phosphopeptides from purified flagella and analyzed them by mass spectrometry. One hundred forty-one phosphorylated peptides were identified, belonging to 32 flagellar proteins. Thereby, 126 in vivo phosphorylation sites were determined. The flagellar phosphoproteome includes different structural and motor proteins, kinases, proteins with protein interaction domains, and many proteins whose functions are still unknown. In several cases, a dynamic phosphorylation pattern and clustering of phosphorylation sites were found, indicating a complex physiological status and specific control by reversible protein phosphorylation in the flagellum.

During ciliogenesis, centrioles convert to membrane-docked basal bodies, which initiate the formation of cilia/flagella and template the nine doublet microtubules of the flagellar axoneme. The discovery that many human diseases and developmental disorders result from defects in flagella has fueled a strong interest in the analysis of flagellar assembly. Here, we will review the structure, function, and development of basal bodies in the unicellular green alga Chlamydomonas reinhardtii, a widely used model for the analysis of basal bodies and flagella. Intraflagellar transport (IFT), a flagella-specific protein shuttle critical for ciliogenesis, was first described in C. reinhardtii. A focus of this review will be on the role of the basal bodies in organizing the IFT machinery.

Abstract Chlamydomonas monoica undergoes homothallic sexual reproduction in response to nitrogen starvation. Mating pairs are established in clonal culture via flagellar agglutination and fuse by way of activated mating structures to form the quadriflagellate zygote. The zygote further matures into a dormant diploid zygospore through a series of events that we collectively refer to as zygosporulation. Mutants that arrest development prior to the completion of zygosporulation have been obtained through the use of a variety of mutagens, including ultraviolet irradiation, 5-fluorodeoxyuridine, ethyl methanesulfonate, and methyl methanesulfonate. Complementation analysis indicates that the present mutant collection includes alleles affecting 46 distinct zygote-specific functions. The frequency with which alleles at previously defined loci have been recovered in the most recent mutant searches suggests that as many as 30 additional zygote-specific loci may still remain to be identified. Nevertheless, the present collection should provide a powerful base for ultrastructural, biochemical, and molecular analysis of zygospore morphogenesis and dormancy in Chlamydomonas.

Cilia and flagella are dynamic organelles of the eukaryotic cell that undergo cycles of assembly/disassembly, in a manner that is coordinated in time with the cell cycle. Cilia are composed by more than 600 peptides and their turnover occurs at the plus distal end of the axoneme; since these organelles lack the machinery required for protein synthesis, a bidirectional transport process known as the IntraFlagellar Transport (IFT) is required to provide flagellar precursors and remove turnover products. IFT is carried on by macromolecular complexes (the IFT particles) which are arranged in polymers (the IFT trains) in the space between the microtubular doublets and the flagellar membrane. IFT trains operate as platforms for cargoes and are moved bidirectionally by specific molecular motors, kinesin-2 as the anterograde motor and dynein-1b as the retrograde motor. Anterograde and retrograde IFT trains possess distinct architectures but, up to now, a high-resolution 3D-model is available only for the anterograde trains, while much less is known on the ultrastructure of retrograde trains. At the distal tip of the organelle, the anterograde transport (from the cell body up to the ciliary distal end or tip) is converted into the retrograde transport (from the tip back to the cell body). Such a turnaround process is strictly required for the correct functioning of the IFT process. So far, however, very little is known about the morpho-functional organization of the tip district, where IFT turnaround takes place. In particular, nothing is known on the interactions that might occur between IFT proteins and the distal tip structures. This doctoral work has been aimed at contributing new information for the comprehension of the IFT turnaround process in the model organism Chlamydomonas reinhardtii. We started our studies from the observation that thin sections of flat-embedded flagella often show anterograde IFT trains that contact the distal end of the central pair microtubules (CP), suggesting the direct involvement of CP capping structures (terminal plates and the ring above) in the IFT turnaround process. We confirmed the interaction of anterograde trains with the CP distal end by electron-tomographic reconstruction of flat-embedded flagellar tips. This approach revealed that anterograde trains split into three components after having reached the end of the A tubule, with the outer part of the train that remains associated with the membrane, the inner part, closer to the microtubule surface, that continues its travel and bends to contact the CP plates, and an intermediate part that stops before reaching the tip. The latter region was interpreted as the part of the train consisting of inactive dynein-1b, which is known to dissociate from the anterograde train before its activation and recruitment for the retrograde transport. Then, we sought to obtain further information on the ultrastructural organization of the distal CP segment. We were able to identify a ladder-like structure (LLS) which is distinctive of this region, is intercalated between the two CP tubules, and is resistant to the cold treatments used to depolymerize tubulin. In order to confirm the association between IFT anterograde trains and the capping CP structures, whole cells were treated with inhibitors of Ca++-dependent protein kinases before flagellar demembranation and negative staining. These inhibitors block the release of kinesin-2 from the anterograde trains and, consequently, IFT turnaround at the tip. As expected, we observed a massive accumulation of IFT particles around the CP terminus. Successively, we analyzed by immunoelectronmicroscopy the specific distribution of the three protein complexes present within the IFT particles. At this purpose, we carried out a series of immunolabeling experiments on grid-absorbed demembranated cells or on sections of resin-embedded samples, using antibodies directed against subunits of the IFT-A complex (IFT139), and of the two IFT-B subcomplexes IFT-B1 (IFT74 and IFT81) and IFT-B2 (IFT172 and IFT57). Our findings suggest that at the tip the IFT-A complex is closely associated with the membrane. On the contrary, both IFT-B1 and IFT-B2 antibodies labelled the distal CP region, though, interestingly, with distinct spatial distributions. IFT-B2 labeling was restricted to approximately the distal 200 nm-segment of the CP, which contains the LLS, and gold particles were never found more distally, above the terminal plates, while IFT-B1 labeling extend also to the ring. The whole set of immunoelectronmicroscopy data indicates that the IFT-B1 and IFT-B2 subcomplexes differentially interact with the distal CP region and its capping structures, and suggests that the IFT-B1 subcomplex might be a main component of the CP capping structures. Accordingly, in our negatively stained samples the cap was shown to consist of thin elongated elements, frequently with a sort of small knob at their mid region; these elements fit quite well with the available IFT-B 3D model. The possibility that IFT-B1 proteins are involved in the formation of the CP cap was confirmed by the analysis of a series Chlamydomonas mutants with defective IFT, which related the presence of the CP cap to the establishment of a fully cycling IFT process. Our data sustain a model of IFT turnaround in which i) the IFT-A complex turns around quickly, remaining associated with the membrane, ii) IFT-B1 and IFT-B2 follow a more complex pathway, during which they separate and differentially interact with the CP distal segment, iii) IFT-B1 directly contribute to the formation of the CP cap. The LLS component, which is ectopically assembled also in mutant strains devoid of the CP tubules, is likely to act as an anchoring structure for IFT-B2 during IFT turnaround.

Subject of study: Cilia and flagella are slender appendages of eukaryotic cells. They are actively bending structures and display regular bending waves. These active flagellar bending waves drive fluid flows on cell surfaces like in the case of the ciliated trachea or propels single cell micro-swimmers like sperm or alga. Objective: The axoneme is the evolutionarily conserved mechanical apparatus within cilia and flagella. It is comprised of a cylindrical arrangement of microtubule doublets, which are the elastic elements and dyneins, which are the force generating elements in the axonemal structure. Dyneins collectively bend the axoneme and must be specifically regulated to generate symmetric and highly asymmetric waveforms. In this thesis, I address the question of the molecular origin of the asymmetric waveform and test different theoretical descriptions for motor regulation. Approach: I introduce the isolated and reactivated Chlamydomonas axoneme as an experimental model for the symmetric and asymmetric flagellar beat. This system allows to study the beat in a controlled and cell free environment. I use high-speed microscopy to record shapes with high spatial and temporal resolution. Through image analysis and shape parameterization I extract a minimal set of parameters that describe the flagellar waveform. Using Chlamydomonas, I make use of different structural and motor mutants to study their effect on the shape in different reactivation conditions. Although the isolated axoneme system has many advantages compared to the cell-bound flagellum, to my knowledge, it has not been characterized yet. Results: I present a shape parameterization of the asymmetric beat using Fourier decomposition methods and find, that the asymmetric waveform can be understood as a sinusoidal beat around a circular arc. This reveals the similarities of the two different beat types: the symmetric and the asymmetric beat. I investigate the origin of the beat-asymmetry and find evidence for a specific dynein motor to be responsible for the asymmetry. I furthermore find experimental evidence for a strong sliding restriction at the basal end of the axoneme, which is important to establish a static bend. In collaboration with P. Sartori and F. Jülicher, I compare theoretical descriptions of different motor control mechanisms and find that a curvature controlled motor-regulation mechanism describes the experimental data best. We furthermore find, that in the dynamic case an additional sliding restriction at the base is unnecessary. By comparing the waveforms of intact cells and isolated reactivated axonemes, I reveal the effect of hydrodynamic loading, and the influence of boundary conditions on the shape of the beating flagella.

Subject of study: Cilia and flagella are slender appendages of eukaryotic cells. They are actively bending structures and display regular bending waves. These active flagellar bending waves drive fluid flows on cell surfaces like in the case of the ciliated trachea or propels single cell micro-swimmers like sperm or alga. Objective: The axoneme is the evolutionarily conserved mechanical apparatus within cilia and flagella. It is comprised of a cylindrical arrangement of microtubule doublets, which are the elastic elements and dyneins, which are the force generating elements in the axonemal structure. Dyneins collectively bend the axoneme and must be specifically regulated to generate symmetric and highly asymmetric waveforms. In this thesis, I address the question of the molecular origin of the asymmetric waveform and test different theoretical descriptions for motor regulation. Approach: I introduce the isolated and reactivated Chlamydomonas axoneme as an experimental model for the symmetric and asymmetric flagellar beat. This system allows to study the beat in a controlled and cell free environment. I use high-speed microscopy to record shapes with high spatial and temporal resolution. Through image analysis and shape parameterization I extract a minimal set of parameters that describe the flagellar waveform. Using Chlamydomonas, I make use of different structural and motor mutants to study their effect on the shape in different reactivation conditions. Although the isolated axoneme system has many advantages compared to the cell-bound flagellum, to my knowledge, it has not been characterized yet. Results: I present a shape parameterization of the asymmetric beat using Fourier decomposition methods and find, that the asymmetric waveform can be understood as a sinusoidal beat around a circular arc. This reveals the similarities of the two different beat types: the symmetric and the asymmetric beat. I investigate the origin of the beat-asymmetry and find evidence for a specific dynein motor to be responsible for the asymmetry. I furthermore find experimental evidence for a strong sliding restriction at the basal end of the axoneme, which is important to establish a static bend. In collaboration with P. Sartori and F. Jülicher, I compare theoretical descriptions of different motor control mechanisms and find that a curvature controlled motor-regulation mechanism describes the experimental data best. We furthermore find, that in the dynamic case an additional sliding restriction at the base is unnecessary. By comparing the waveforms of intact cells and isolated reactivated axonemes, I reveal the effect of hydrodynamic loading, and the influence of boundary conditions on the shape of the beating flagella.:Contents 1 Introduction. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.1 Biology of Cilia and Flagella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.1.1 The dimensions of flagellated micro-swimmers . . . . . . . . . . . . . . . . . 4 1.1.2 The symmetric and the asymmetric beat . . . . . . . . .. . . . . . . . . . . . 5 1.1.3 Chlamydomonas reinhardtii as a flagella model . . . . . . . . . . 5 1.2 The axoneme is the internal structure in eukaryotic cilia and flagella . . 6 1.3 Structure and function of microtubules and dyneins . . . . . . . . . . . 9 1.3.1 Microtubules: The structural elements in the axoneme . . . . . . 9 1.3.2 Dyneins: The force generators that drive the axonemal beat . . . 10 1.3.3 The asymmetries in the axoneme and consequences for the beat 17 1.4 Axonemal waveform models and mechanisms: from sliding to bending to beating . . . . . . . . . . . . . . 20 1.5 Geometrical representation and parameterization of the axonemal beat . . . . . . . . . . . . . . . 23 2 Questions addressed in this thesis . . . . . . . . . . . . . . 27 3 Material and Methods . . . . . . . . . . . . . . 29 3.1 Chlamydomonas cells: Axoneme preparation and motility assays . . . . 29 3.1.1 Culturing of Chlamydomonas reinhardtii cells . . . . . . . . . . . 29 3.1.2 Isolation, demembranation and storage of axonemes . . . . . . . 33 3.1.3 Reactivation of axonemes in controlled conditions . . . . . . . . . 35 3.1.4 Axoneme gliding assay using kinesin 1 . . . . . . . . . . . . . . . 36 3.2 Imaging and image processing . . . . . . . . . . . . . . . . . . . . . . . . 38 3.2.1 High-speed imaging of the flagella and axonemes . . . . . . . . . 38 3.2.2 Precise tracking of isolated axonemes and the flagella of cells . . 42 3.2.3 High throughput frequency evaluation of isolated axonemes . . . 47 3.2.4 Beat frequency characterization of the reactivated WT axoneme . . . . . . . . . . . . . . 49 4 Results and Discussion . . . . . . . . . . . . . . 53 4.1 The beat of the axoneme propagates from base to tip . . . . . . . . . . . 53 4.1.1 TEM study reveals no sliding at the base of a bend axoneme . . 53 4.1.2 The direction of wave propagation is directly determined from the reactivation of polarity marked axonemes . . . . . . . . . . 57 4.1.3 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 4.2 The asymmetric beat is the superposition of a static semi-circular arc and a sinusoidal beat . . . . . . .. . . . . . . . . . . . . . . . . 61 4.2.1 The waveform is parameterized by Fourier decomposition in time . . . . . . . . . . . . . . 61 4.2.2 The 0th and 1st Fourier modes describe the axonemal waveform . . . . . . . . . . . . . . 65 4.2.3 General dependence of shape parameters on axoneme length and beat frequency. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 4.2.4 The isolated axoneme is a good model for the intact flagellum . .. . . . . . . . . . . . . . 71 4.2.5 Summary: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 4.3 The circular motion is a consequence of the axonemal waveform . . . . . . . . . . . . . . . . . . . 79 4.3.1 Hydrodynamic relations for small amplitude waves explain the relation between swimming and shape of axonemes . . . . 79 4.3.2 The swimming path can be reconstructed using shape information and a hydrodynamic model . . . . . . . . . . . . . . . . 83 4.3.3 Motor mutations alter the direction of rotation of reactivated axonemes. . . . . . . . . . . . . . . . . . . . . . . . 84 4.3.4 Summary: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 4.4 The molecular origin of the circular mean shape. . . . . . . . . . . . . . 89 4.4.1 Motor Mutations do not abolish the mean shape, a structural mutation does . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 4.4.2 The axoneme is straight in absence of ATP but bend at low ATP concentrations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 4.4.3 Viscous load decreases the mean curvature . . . . . . . . . . . . 99 4.4.4 Summary: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 4.5 Curvature-dependent dynein activation accounts for the shape of the beat of the isolated axoneme . . . . . . . . . . . . . . . . 103 5 Conclusions and Outlook . . . . . . . . . . . . . . . . 109 5.1 Summary of the results . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 5.2 Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 Abbreviations . . . . . . . . . . . . . . . . 111 List of figures . . . . . . . . . . . . . . . . 116 List of tables . . . . . . . . . . . . . . . . 118 Bibliography

Soft X-ray contact microscopy (SXCM), has many applications in both life and material sciences. In the case of life sciences SXCM enables the study of the ultrastructure of living hydrated specimens, without the need of dehydration or other chemical pretreatment, by using suitable pulsed X-ray sources such as laser plasmas [1,2]. The interest in using soft X-rays, in the so called “water window” (2.3-4.4nm), is based on the low attenuation at these wavelengths caused by water, as compared to the attenuation caused by organic matter. Therefore, good contrast masking of the incident radiation is provided. The successful imaging of biological specimen, requires the development of sensitive photoresist materials for image recording; these should have capabilities of high resolution lithography and an extended grayscale. Up to now, the only known photoresist used successfully in SXCM has been polymethyl methacrylate (PMMA). This is a high resolution photoresist when exposed to e-beam or X-ray radiation, with contrast suitable for gray scale recording ; nevertheless, it is a relatively slow photoresist and, therefore, requires a very large fluence of X-rays for imaging. The work reported here was carried out using the Vulcan Nd:glass laser facility at the Rutherford Appleton Laboratory UK, whose rod chain output can deliver more than 11 J at 1064nm. This can be delivered on a Yttrium target as an X- ray source. A very sensitive e -- beam photoresist, used for the first time in SXCM, enabled the biological imaging with the specific source in single pulse experiments in the water window spectral range. This photoresist is an epoxy novolac based chemically amplified photoresist (EPR) which has been proven capable of resolving sub tenth micron features. Initial experiments to compare the sensitivity of PMMA and EPR were done in the absence of a biological specimen. The first image for EPR is obtained at 300mJ laser pulse energy. PMMA used as a reference gave a first image of 40nm depth difference between exposed and unexposed areas, as measured with a Dektak profilometer, at 4.6 J laser pulse energy and an image of 70 nm at 10.6 J. Thus on the basis of the calculations of X-ray flux produced at different laser energies, the minimum flux for image production with PMMA is 2.5mJ.cm-2 and the corresponding value for EPR is only ~ 0.07 mJ.cm-2, giving a difference of two orders of magnitude approximately, for the two materials. In biological imaging experiments the living specimens were cells of the motile green alga, Chlamydomonas, which were placed in a droplet of medium. In the experiments with biological specimens no image was obtained with PMMA as a recording material, even with the higher pulse energy available and careful adjustment of water layer thickness in order to be exactly the size of cell diameter. In the contrary, with the EPR resist biological imaging was possible. Images of Chlamydomonas cells were successfully obtained. These images clearly show the cell body and the flagella, suggest a lateral resolution considerably better than 300nm.