L. Mir

The structure of the flagellar apparatus of the swarm cells ofPhysarum polycephalum

SummaryFlagellation ofPhysarum polycephalum amoebae (Myxomycete) involves the formation around the two kinetosomes of a flagellar apparatus leading to a modification in the shape of the amoeba and its nucleus. A tridimensional ultrastructural model of the flagellar apparatus is proposed, based upon observation of the isolated nucleo-flagellar apparatus complex. The flagellar apparatus is composed of a non-microtubular structure (the posterior para-kinetosomal structure), five microtubular arrays and two flagella: a long anterior flagellum and a short flagellum directed backwards. The asymmetry of the flagellar apparatus is due mainly to the presence of the posterior para-kinetosomal structure on the right side of the posterior kinetosome and of the two asymmetrical microtubular arrays 3 and 4. Thus, the flagellar apparatus is right-handed. This asymmetry implies also some spatial constraints on two other microtubular arrays (2 and 5). Except in the case of the microtubular array 1 which links the proximal end of the anterior kinetosome to the nuclear membrane, the number of microtubules of each microtubular array seems to be well defined: 39, 5–6, 7–9, and 2+2 for the microtubular arrays 2, 3, 4, and 5 respectively. All the elements of the nucleo-flagellar apparatus complex are linked either directly or indirectly through bridges. Furthermore, the microtubules which composed the microtubular array 3 are linked through bridges while the microtubules of the microtubular arrays 2, 3, and 4 seem to be linked through a reticulate material. All these spatial relationships lead to a great cohesion of the nucleo-flagellar apparatus complex which appears to be a well defined structure. This suggests thatPhysarum amoebal flagellation can be a promising system to study the morphogenesis of an eucaryotic cell.

The structure of the pro-flagellar apparatus of the amoebae ofPhysarum polycephalum: Relationship to the flagellar apparatus

SummaryUnflagellated amoebae ofPhysarum polycephalum (Myxomycete) possess a pro-flagellar apparatus. A tridimensional ultrastructural model of the pro-flagellar apparatus is proposed, based upon observations of thin sections of whole amoebae and of isolated nucleo-pro-flagellar apparatus complexes. The pro-flagellar apparatus is composed by the same basic elements as the fully differentiated flagellar apparatus and differs from the latter by three main aspects: the two kinetosomes of the pro-flagellar apparatus are devoid of flagella; the posterior kinetosome is directed forward, and, with the exception of microtubular arrays 1 and 5, all the other microtubular arrays (2–4) have a reduced length, although they show a normal number of constituting microtubules. We demonstrate the existence of an extension of the posterior para-kinetosomal structure which is present both in the pro-flagellar and in the flagellar apparatus. The growth of the microtubules of the pericentriolar arrays and of the flagella could be the main event which leads to the formation of the fully developed flagellar apparatus inPhysarum amoebae.

Centriole maturation in the amoebae ofPhysarum polycephalum

SummaryThe precise geometry of pro-centriole formation has been studied inPhysarum polycephalum amoebae. The spatial references used were the posterior and the anterior kinetosomes which are unequivocally defined by the presence of the posterior para-kinetosomal structure, the microtubular array 4 and the microtubular arrays 1, 2, and 3. The observations made suggest that pro-centrioles follow a maturation process. A pro-centriole formed during the nth cell cycle becomes the posterior kinetosome during the (n + 1)th cell cycle and the anterior one during all the following cell cycles. Pro-centriole formation occurs late in the cell cycle. This observation disagrees with a role of pro-centriole formation in the regulation of S phase in contrast to what has been suggested in other eucaryotic cells.

Microtubule cytoskeleton and morphogenesis in the amoebae of the myxomycete Physarum polycephalum

Summary— The amoebae of the myxomycete Physarum polycephalum are of interest in order to analyze the morphogenesis of the microtubule and microfilament cytoskeleton during cell cycle and flagellation. The amoebal interphase microtubule cytoskeleton consists of 2 distinct levels of organization, which correspond to different physiological roles. The first level is composed of the 2 kinetosomes or centrioles and their associated structures. The anterior and posterior kinetosomes forming the anterior and posterior flagella are morphologically distinguishable. Each centriole plays a role in the morphogenesis of its associated satellites and specific microtubule arrays. The 2 distinct centrioles correspond to the 2 successive maturation stages of the pro‐centrioles which are built during prophase. The second level of organization consists of a prominent microtubule organizing center (mtoc 1) to which the anterior centriole is attached at least during interphase. This mtoc plays a role in the formation of the mitotic pole. These observations based on ultrastructural and physiological analyses of the amoebal cystoskeleton are now being extended to the biochemical level. The complex formed by the 2 centrioles and the mtoc 1 has been purified without modifying the microtubule‐nucleating activity of the mtoc 1. Several microtubule‐associated proteins have been characterized by their ability to bind taxol‐stabilized microtubules. Their functions (e.g., microtubule assembly, protection of microtubules against dilution or cold treatment, phosphorylating and ATPase activities) are under investigation. These biochemical approaches could allow in vitro analysis of the morphogenesis of the amoebal microtubule cytoskeleton.

Variations in the number of centrioles, the number of microtubule organizing centers 1 and the percentage of mitotic abnormalities inPhysarum polycephalum amoebae

SummarySeveral, stable amoebal strains which differ phenotypically from the diploid parental amoebal strain have been obtained in the MyxomycetePhysarum polycephalum. They were detected using their flagellation pattern as a discriminating parameter. This approach is valid since the number of flagella by phase contrast microscopy correlates with the number of anterior centrioles obtained using three-dimensional reconstructions of the nucleo-flagellar complexes from serial thin sections. The complexity of the structures of the various nucleo-flagellar complexes suggests that in these strains the duplication time of centrioles is not strictly regulated as it is in haploid amoebae. In agreement with this hypothesis, several pro-centrioles were observed in interphase amoebae. Although the anterior centrioles are linked to the mtoc 1 during interphase, the number of mtoc 1 cannot regulate the number of centrioles since some strains possess two mtoc 1 but only one pair of centrioles. Neither the number of centrioles nor the number of mtoc 1 are related to ploidy. Stable strains with one (all haploid strains), two (some diploid strains) and three (some diploid strains) mtoc 1 have been observed. Thus each mtoc 1 is duplicated once per cell cycle implying that it must possess some information which plays a role in the morphogenesis of the new mtoc 1. Except in one case, the number of mitotic abnormalities increases exponentially with the number of mtoc 1. This observation suggests that the mtoc 1 could correspond to the interphase state of the mitotic center.

A technique to design complex 3D lab on a chip involving multilayered fluidics, embedded thick electrodes and hard packaging—application to dielectrophoresis and electroporation of cells

Nowadays, lab on chips (LOCs) require the development of new technologies in order to integrate complex fluidics, sensors, actuators, etc. Such integration requires overcoming both technological bottlenecks and an increase in production cost. We propose a technique to manufacture reusable and complex LOCs made up of SU-8 resist for the fluidic structure and of glass for the hard packaging, and are compatible with the integration of thick electrodes. The method is based on the combination of two bonding technologies, both based on a wafer bonder. The first one consists of the bonding of a thin photosensitive SU-8 dry film, which is similar to lamination. The second one is the standard bonding technique which uses a hard substrate covered by an SU-8 layer. The LOCs that can be obtained by combining these two methods are transparent, and include 3D microfluidic structures and thick electrodes. Moreover, these LOCs are reusable, packaged and ready to use. In order to validate the concept, we designed an LOC devoted to cell arraying, using dielectrophoresis, as well as to cell electroporation.

Unusual amoebal strains of the MyxomycetePhysarum polycephalum possessing two pro-flagellar apparatuses

SummaryThe microtubules structure of two stable diploid amoebal strains, each resulting from the fusion of two haploid amoebae has been studied by electron microscopy. Tridimensional reconstructions showed that these diploid amoebae-typically possessed two proflagellar apparatuses,i.e., two microtubule organizing centers 1 (mtoc 1) and two pairs of centrioles with their associated microtubular arrays. These observations account for the high frequency of biflagellated amoebae in these two strains. The presence of two mtoc 1 may account for the high percentage of mitotic abnormalities which was observed under phase contrast microscopy and electron microscopy and is in agreement with a role of the mtoc 1 as a mitotic center during mitosis. However, the presence of numerous normal mitotic apparatuses raises the question of the regulations which play a role in the mitotic process. The unusual distribution of centrioles and the unusual pro-flagellar apparatuses which were produced suggest that in interphase the anterior centriole is a necessary structure for the morphogenesis of the microtubular arrays 2 and 3 and that the posterior centriole is a necessary structure for the morphogenesis of the microtubular arrays 4 and 5.

A microfluidic device with removable packaging for the real time visualisation of intracellular effects of nanosecond electrical pulses on adherent cells

The biological mechanisms induced by the application of nanosecond pulsed electric fields (nsPEFs: high electrical field amplitude during very short duration) on cells remain partly misunderstood. In this context, there is an increasing need for tools that allow the delivering of such pulses with the possibility to monitor their effects in real-time. Thanks to miniaturization and technology capabilities, microtechnologies offer great potential to address this issue. We report here the design and fabrication of a microfluidic device optimized for the delivery of ultra short (10 ns) and intense (up to 280 kV cm(-1)) electrical pulses on adherent cells, and the real time monitoring of their intracellular effects. Ultra short electric field pulses (nsPEFs or nanopulses) affect both the cell membrane and the intracellular organelles of the cells. In particular, intracellular release of calcium from the endoplasmic reticulum was detected in real time using the device, after exposure of adherent cells to these nsPEFs. The high intensity and spatial homogeneity of the electric field could be achieved in the device thanks to the miniaturization and the use of thick (25 μm) electroplated electrodes, disposed on a quartz substrate whose transparency allowed real time monitoring of the nsPEFs effects. The proposed biochip is compatible with cell culture glass slides that can be placed on the chip after separate culture of several days prior to exposure. This device allows the easy exposure of almost any kind of attached cells and the monitoring in real time while exposed to nsPEFs, opening large possibilities for potential use of the developed biochips.

Biomedical Applications of Short, Intense Electric Pulses

The biomedical applications of electric pulses in biology and medicine are, at the same time, an old and a new topic. Indeed, the use of low intensity electric pulses for long durations is known and used from a long time. Functional electrical stimulation, antalgic treatments as well as wound healing acceleration are clinical applications of that type of electric pulses. Nowadays, it has become increasingly clear that there are also interesting biomedical applications of the use of short, intense electric pulses delivered for very short times. These electric pulses result in changes of cell membrane properties that have been termed electropermeabilization or electroporation. Several potential applications of these electric pulses, delivered either ex vivo or in vivo, have been described in preclinical studies. Two of these applications are currently being tested in clinical trials. This article focuses on the basic concepts and the recent clinical developments of the biomedical applications of short, intense electric pulses delivered for very short periods.