Pawel Pasierbek

• PhD
• Microscopy Specialist at Institute of Molecular Biotechnology

Operating shared resource laboratories (SRL) in times of pandemic is a challenge for research institutions. In a multi‐user, high‐turnover working space, the transmission of infectious agents is difficult to control. To address this challenge, imaging core facility managers being members of German BioImaging discussed how shared microscopes could b...

Tissue clearing combined with deep imaging has emerged as a powerful alternative to classical histological techniques. Whereas current techniques have been optimized for imaging selected nonpigmented organs such as the mammalian brain, natural pigmentation remains challenging for most other biological specimens of larger volume. We have developed a...

High refractive index organic solvents are commonly used as an imaging medium in tissue clearing approaches. While effective, such solvents provide serious concerns for the safety of users and the equipment, especially in a central microscopy unit. To overcome these concerns, we have developed a large and re-usable imaging chamber compatible with t...

Turbidity and opaqueness are inherent properties of tissues that limit the capacity to acquire microscopic images through large tissues. Creating a uniform refractive index, known as tissue clearing, overcomes most of these issues. These methods have enabled researchers to image large and complex 3D structures with unprecedented depth and resolutio...

Turbidity and opaqueness are inherent properties of tissues which limit the capacity to acquire microscopic images through large tissues. Creating a uniform refractive index, known as tissue clearing, overcomes most of these issues. These methods have enabled researchers to image large and complex 3D structures with unprecedented depth and resoluti...

Autophagy is a mechanism by which starving cells can control their energy requirements and metabolic states, thus facilitating the survival of cells in stressful environments, in particular in the pathogenesis of cancer. Here we report that tissue-specific inactivation of Atg5, essential for the formation of autophagosomes, markedly impairs the pro...

Time lapse series of SUN-1(S12E)::GFP in the sun-1(ok1282) background in the distal part of the prolonged TZ. The three insets show three enlarged independent nuclei. Scale bar: 2 µm. (0.98 MB MOV)

Time lapse series of SUN-1(S12E)::GFP in the sun-1(ok1282) background in the proximal part of the prolonged TZ. The three insets show three enlarged independent nuclei. Scale bar: 2 µm. (0.98 MB MOV)

Time lapse series of SUN-1::GFP in the cra-1(tm2144) background. The three insets show three enlarged independent nuclei. Scale bar: 2 µm. (0.98 MB MOV)

Time lapse series of SUN-1::GFP with counterstaining of the chromatin using Hoechst 33342. The first inset shows the merging of SUN-1::GFP (green) and the chromatin (blue). The second inset shows the chromatin and the third inset SUN-1::GFP. Scale bar: 2 µm. (1.16 MB MOV)

Time lapse series of SUN-1::GFP in the spo-11(me44) background; irradiated 2-d-old hermaphrodites. The three insets show three enlarged independent nuclei. Scale bar: 2 µm. (0.84 MB MOV)

Time lapse series of SUN-1::GFP in the spo-11(me44) background. The three insets show three enlarged independent nuclei. Scale bar: 2 µm. (0.84 MB MOV)

Time lapse series of SUN-1::GFP in the sun-1(ok1282) background; irradiated 2-d-old hermaphrodites. The three insets show three enlarged independent nuclei. Scale bar: 2 µm. (0.84 MB MOV)

Long Noncoding RNAs The past 5 years have uncovered thousands of long (>100 nucleotides) noncoding RNAs (lncRNAs) outpacing our understanding of their functions and mechanisms in regulating the genome. Lee (p. 1435 ) reviews the known and suspected means by which these intriguing molecules control gene expression locally and at great distances, con...

Background movement during time-lapse microscopy and definition of Maxwellian-shaped distribution. (A) Worms were killed in sodium azide and analyzed. Each line corresponds to the distribution of the projected speed of SUN-1::GFP aggregates in wild-type (blue), him-3(gk149) (yellow), and htp-1(gk174) (red) backgrounds. The corresponding cumulative...

Time lapse series of SUN-1::GFP in the htp-1(gk174) background. The three insets show three enlarged independent nuclei. Scale bar: 2 µm. (0.26 MB MOV)

Lateral elements are required for proper loading of PC proteins. Localization of the PC protein ZIM-3 in him-3(gk149) and htp-1(gk174). Immunostaining of ZIM-3 (red) and SUN-1::GFP (green); DAPI (blue). (1.96 MB TIF)

Fisher's exact test to assess the difference between wild type and all other genotypes tested for the values ‘number of fusion/splitting events’. Significant p-values (p<0.05) are highlighted in bold. (0.06 MB DOC)

Supplemental methods. (0.03 MB DOC)

Time lapse series of SUN-1(G311V)::GFP in the sun-1(ok1282) background. The three insets show three enlarged independent nuclei. Scale bar: 2 µm. (0.84 MB MOV)

Dynamics of SUN-1(S12E)::GFP aggregates. (A) Numbers of aggregates in the regions proximal to the mitotic zone. The variations indicated correspond to the standard deviation. (B) Number of SUN-1 fusion/splitting events grouped into classes. (C) Quantification of the coalescence time (t) grouped into classes (t<1 min, 1 min≤t<3 min, and t≥3 min). (D...

Correlation between speed and size of SUN-1 aggregates. (A) A deconvolved image was converted to a binary image using a threshold and then initial segmentation restored using a watershed transform (i). Aggregates <2.15 µm2 were defined as foci, aggregates >2.15 µm2 as patches. Output image (yellow) overlaid with the starting picture (i, right panel...

Fisher's exact to assess the difference between wild type and all other genotypes tested for the values' time-window of SUN-1 aggregate coalescence'. Significant p-values (p<0.05) are highlighted in bold. (0.05 MB DOC)

Time lapse series of SUN-1::GFP in the him-3(gk149) background. The three insets show three enlarged independent nuclei. Scale bar: 2 µm. (0.56 MB MOV)

Time lapse series of SUN-1::GFP in the syp-2(ok307) background; proximal part of the TZ. The three insets show three enlarged independent nuclei. Scale bar: 2 µm. (0.55 MB MOV)

Time lapse series of SUN-1::GFP in the prom-1(ok1140) background. The three insets show three enlarged independent nuclei. Scale bar: 2 µm. (0.84 MB MOV)

Arc computation. Distances a and b were calculated from the most extreme positions of the tracks (orange in A [SUN-1::GFP], red in A' [SUN-1(G311V)::GFP]). Distance c was calculated using the Pythagoras theorem (B, B'). c was circumscribed on a circle with the radius of the average size of a nucleus. Angle β (pink) was calculated using the cosine l...

Lack of patterns for the traveled distance of SUN-1::GFP aggregates in nuclei located at different positions in the TZ. Box plot of the arc values for subregions in TZ (distal, central, and proximal parts of TZ). Red box plot, first movie; blue box plot, second movie. Green line represents the median value in the distribution of the arc; extremitie...

SUN-1 aggregates are present in nuclei with loose clustering of the chromatin. Immunostaining of SUN-1 in wild type, htp-1(gk174); syp-1(RNAi) and cra-1(tm2144). White arrows indicate nuclei with loose clustering of the chromatin. (3.82 MB TIF)

An excess of DSBs affected pairing but not SC polymerization. Dissected gonads of aged irradiated and nonirradiated worms were divided into seven zones of equal length (A), and the pairing of homologs was assessed by FISH with a probe for 5S rDNA (on chromosome V). (B) Pairing in nonirradiated 3-d-old wild-type gonads (dark blue) and three-d-old wi...

Effect of SUN-1 phosphorylation on aggregate dynamics. Projection of the cumulative movement of SUN-1(S12E)::GFP (A, A'), displacement tracks (B, B'), distribution of the projected speed (C, C'), and arcs (D, D'). Blue lines represent values from the first movie; orange lines values from the second. (A, B, C, D) from distal TZ, (A', B', C', D') fro...

Supplemental results. (0.04 MB DOC)

Time lapse series of SUN-1::GFP in the sun-1(ok1282) background. The three insets show three enlarged independent nuclei. Scale bar: 2 µm. (0.76 MB MOV)

Time lapse series of SUN-1::GFP in the syp-2(ok307) background; distal part of the TZ. The three insets show three enlarged independent nuclei. Scale bar: 2 µm. (0.55 MB MOV)

Time lapse series of SUN-1::GFP in the syp-3(me42) background. The three insets show three enlarged independent nuclei. Scale bar: 2 µm. (0.55 MB MOV)

Time lapse series of SUN-1::GFP in the htp-1(gk174); syp-1(RNAi) background in the distal part of the zone with SUN-1 aggregates. The three insets show three enlarged independent nuclei. Scale bar: 2 µm. (0.87 MB MOV)

Time lapse series of SUN-1::GFP in the him-19(jf6) background; non-irradiated 2-d-old hermaphrodites. Scale bar: 2 µm. (0.96 MB MOV)

Time lapse series of SUN-1::GFP in the htp-1(gk174); syp-1(RNAi) background in the proximal part of the zone with SUN-1 aggregates. The three insets show three enlarged independent nuclei. Scale bar: 2 µm. (0.84 MB MOV)

Author Summary During meiosis, homologous chromosomes from each parent must pair, synapse, and recombine before being assorted to the gametes. In Caenorhabditis elegans, to find the correct pairing partner, telomere-led chromosome movement occurs in a restricted subvolume of the nucleus. This feature is comparable to the widely conserved meiotic bo...

Recognition of pathogens by the innate immune system requires proteins that detect conserved molecular patterns. Nucleic acids are recognized by cytoplasmic sensors as well as by endosomal Toll-like receptors (TLRs). It has become evident that TLRs require additional proteins to be activated by their respective ligands. In this study, we show that...

Genome haploidization during meiosis depends on recognition and association of parental homologous chromosomes. The C. elegans SUN/KASH domain proteins Matefin/SUN-1 and ZYG-12 have a conserved role in this process. They bridge the nuclear envelope, connecting the cytoplasm and the nucleoplasm to transmit forces that allow chromosome movement and h...

Double-stranded RNA (dsRNA)-binding proteins interact with substrate RNAs via dsRNA-binding domains (dsRBDs). Several proteins harboring these domains exhibit nucleocytoplasmic shuttling and possibly remain associated with their substrate RNAs bound in the nucleus during nuclear export. In the human RNA-editing enzyme ADAR1-c, the nuclear localizat...

A novel gene, prom-1, was isolated in a screen for Caenorhabditis elegans mutants with increased apoptosis in the germline. prom-1 encodes an F-box protein with limited homology to the putative human tumor suppressor FBXO47. Mutations in the prom-1 locus cause a strong reduction in bivalent formation, which results in increased embryonic lethality...

Three-dimensional fluorescence microscopy has found widespread applications in recent years, especially in molecular cell biology. Imaging in this type of microscopy is usually based on a series of sectioned images obtained by stepping the specimen through the focal region of a beam type scanning microscope. In most confocal or two-photon microscop...

Condensed Y chromosomes in Rumex acetosa L. root-tip nuclei were studied using 5-azaC treatment and immunohistochemical detection of methylated histones. Although Y chromosomes were decondensed within root meristem in vivo, they became condensed and heteropycnotic in roots cultured in vitro. 5-azacytidine (5-azaC) treatment of cultured roots caused...

The meiotically expressed Zip3 protein is found conserved from Saccharomyces cerevisiae to humans. In baker's yeast, Zip3p has been implicated in synaptonemal complex (SC) formation, while little is known about the protein's function in multicellular organisms. We report here the successful targeted gene disruption of zhp-3 (K02B12.8), the ZIP3 hom...

The nematode Caenorhabditis elegans is one of the genetically best-studied organisms. Its genome was completely sequenced, and, having a determinate cell lineage, the fate of every single cell during embryonic development was uncovered (for review see Riddle et al., 1997). C. elegans is a hermaphrodite with 2n = 12 holocentric chromosomes. Its meio...

The product of the Caenorhabditis elegans ORF F18E2.3 is homologous to the cohesin component Scc3p. By antibody staining the product of F18E2.3 is found in interphase and early meiotic nuclei. At pachytene it localizes to the axes of meiotic chromosomes but is no longer detectable on chromatin later in meiosis or in mitoses. Depletion of the gene p...

We investigated the role of Caenorhabditis elegans rad-51 during meiotic prophase. We showed that rad-51 mutant worms are viable, have no defects in meiotic homology recognition and synapsis but exhibit abnormal chromosomal morphology and univalent formation at diakinesis. During meiosis RAD-51 becomes localized to distinct foci in nuclei of the tr...

Mitotic chromosome segregation depends on bi-orientation and capture of sister kinetochores by microtubules emanating from opposite spindle poles and the near synchronous loss of sister chromatid cohesion. During meiosis I, in contrast, sister kinetochores orient to the same pole, and homologous kinetochores are captured by microtubules emanating f...

Chromosome segregation during mitosis and meiosis is triggered by dissolution of sister chromatid cohesion, which is mediated by the cohesin complex. Mitotic sister chromatid disjunction requires that cohesion be lost along the entire length of chromosomes, whereas homolog segregation at meiosis I only requires loss of cohesion along chromosome arm...

We have studied four Caenorhabditis elegans homologs of the Rad21/Scc1/Rec8 sister-chromatid cohesion protein family. Based on the RNAi phenotype and protein localization, it is concluded that one of them, W02A2.6p, is the likely worm ortholog of yeast Rec8p. The depletion of C. elegans W02A2.6p (called REC-8) by RNAi, induced univalent formation a...

Molecular cytogenetics, particularly the localization of DNA sequences by in situ hybridization, has increased our understanding about the genomic structure of plants and animals. We demonstrate here the application of an improved nonfluorescent in situ hybridization system detection (DAKO GenPoint system) to plant chromosomes. Using this system, h...