Conventional kinesin is a motor protein which translocates organelles from cell centre to cell periphery along specialized filamentous tracks, called microtubules. The direction of translocation is determined by microtubule polarity. This process of biological force generation can be simulated outside cells with kinesin-coated particles actively moving along immobilized microtubules. The in vitro approaches of kinesin-mediated transport described so far had the disadvantage that concerning their polarity the microtubules were randomly distributed resulting in random transport direction. The present paper demonstrates the unidirectional translocation of kinesin-coated cargoes across arrays of microtubules aligned not only in a geometrically parallel but also in an isopolar fashion. As cargo, glass, gold, and polystyrene beads with diameters between 1 and 10 µm were used. Independent of material and size, these beads were observed to move unidirectionally with average velocities of 0.3-1.0 µm s-1 over distances up to 2.2 mm. Moreover, the isopolar microtubule arrays even enabled the transport of large flat glass particles with an area of up to 24 µm×12 µm and 2-5 µm thickness which obviously contacted more than one microtubule. The controlling transport direction is considered to be an essential step for future developments of motor protein-based microdevices working in nanometre steps.
Summary: Microscopical imaging of natural, unstressed draglines or of untreated bulk samples showed two types or threads with diameters of either ~ 1-2 µm or 4-5 µm, which could be identified as products of the minor or major ampullate glands. The threads had a circular profile in serial cross sections and are surrounded by a thin outer layer of a different material within the section. Such fibrillar configurations were also found in untreated threads or in the same serial sections of transmission electron microscopy (TEM) samples by means of the special technique of laser scanning microscopy. In TEM slides, numerous cavities with the same circular profile were detectable, and the length of these cavities is variable from 40-300 nm. The threads are oriented parallel and twisted around themselves to construct a double thread. In the interface between the two single threads, bridge-like structures are prominent. The single untreated thread consists of cylindrical fibers with a diameter of approximately 1-1.5 µm. Apparently more than eight fibers are within a thread and each fiber is composed of a great number of fibrils with a diameter of about 150 nm. The surface of threads is coated with a characteristic layer ~150-250 nm thick that contains glycoproteins. These were demonstrated for the first time by labeling with concanavalin A lectin-gold complex and are dependent on the diameter and length of the thread. The same substances could also be detected inside the single thread. The skin can be removed completely or partially by mechanical treatment, or by washing with phosphatebuffered saline or trypsin.
The processes taking place during routine chromosome preparation are not well understood. In this study, the morphological changes in amniotic fluid cells, blood lymphocytes, and bone marrow cells in the metaphase stage were examined under an inverted microscope during chromosome preparation. The putative processes that occur during chromosome preparation were simulated in suspension, and the cells were treated with different mixtures of hypotonic solution, fixative, methanol, acetic acid, and water. Evaporation of the fixative was performed under normal atmospheric conditions and under vacuum at different levels of humidity. Freeze fracture electron microscopy was used to analyze the effects of fixative on the cell membrane. Confocal microscopic analysis was used to investigate three-dimensionally the effects of hypotonic treatment on the positions of chromosomes in fixed mitotic lymphocytes. Chromosome preparation-induced changes in the lengths of single chromosomes were also investigated. The results show that chromosome spreading involves significant water-induced swelling of mitotic cells during evaporation of the fixative from the slide, which is a prerequisite for chromosomal elongation, the production of metaphase spreads for chromosome analysis, and the appearance of Giemsa banding patterns. Hypotonic treatment is essential for well-spread metaphase chromosomes because it moves the chromosomes from a central to a more peripheral position in the cell, where they can be stretched more effectively during mitotic swelling. Like mitotic cells, isolated single chromosomes also have their own potential to swell and lengthen during chromosome preparation. We hypothesize that chromosome preparation leads to a genome-wide chromosomal region–specific opening of chromatin structures as GTG-light bands and sub-bands. Living cells may possess a similar mechanism, which is used only to open single chromatin structures to facilitate transcription. We propose the concept of chromosomal region–specific protein swelling.
In contrast to those of metaphase chromosomes, the shape, length, and architecture of human interphase chromosomes are not well understood. This is mainly due to technical problems in the visualization of interphase chromosomes in total and of their substructures. We analyzed the structure of chromosomes in interphase nuclei through use of high-resolution multicolor banding (MCB), which paints the total shape of chromosomes and creates a DNA-mediated, chromosome-region-specific, pseudocolored banding pattern at high resolution. A microdissection-derived human chromosome 5-specific MCB probe mixture was hybridized to human lymphocyte interphase nuclei harvested for routine chromosome analysis, as well as to interphase nuclei from HeLa cells arrested at different phases of the cell cycle. The length of the axis of interphase chromosome 5 was determined, and the shape and MCB pattern were compared with those of metaphase chromosomes. We show that, in lymphocytes, the length of the axis of interphase chromosome 5 is comparable to that of a metaphase chromosome at 600-band resolution. Consequently, the concept of chromosome condensation during mitosis has to be reassessed. In addition, chromosome 5 in interphase is not as straight as metaphase chromosomes, being bent and/or folded. The shape and banding pattern of interphase chromosome 5 of lymphocytes and HeLa cells are similar to those of the corresponding metaphase chromosomes at all stages of the cell cycle. The MCB pattern also allows the detection and characterization of chromosome aberrations. This may be of fundamental importance in establishing chromosome analyses in nondividing cells.
In routine chromosome harvesting of blood lymphocytes it is well accepted that metaphase spreads are obtained from fixed mitotic cells which burst on the surface of slides during the dropping procedure. For confirmation and clarification, fixed mitotic cells were dropped onto coverslips and observed under an inverted microscope during the evaporation of the fixative. Fixed mitotic cells in the metaphase stage first stick onto the surface of the coverslip without changing their three-dimensional shape and they do not burst. Thereafter, when evaporation of the fixative occurs, they slowly flatten until they are spread. This slow process leads to a stretching of chromosomes which may be a prerequisite for high resolution banding patterns. Confocal laser scanning microscopic measurements of the length, thickness, and width of chromosomes after (i) short term evaporation of the fixative, (ii) evaporation of the fixative under routine harvesting conditions and (iii) a prolonged evaporation, confirmed the stretching of chromosomes. The humidity, the temperature, and the drying time of the fixative influence the dynamic flow of the remaining fixative on the slide. This dynamic flow leads to an intensive wash of the fixed mitotic cells with increasing concentrations of acetic acid which is primarily responsible for the better quality of the metaphase spread.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.