Attachment Zone Research Articles

Polo-Like Kinase Is Expressed in S/G 2 /M Phase and Associated with the Flagellum Attachment Zone in both Procyclic and Bloodstream Forms of Trypanosoma brucei

Trypanosoma brucei, the etiologic agent of African sleeping sickness, divides into insect (procyclic) and bloodstream forms. These two forms are subject to distinct cell cycle regulations, with cytokinesis controlled primarily by basal body/kinetoplast segregation in the procyclic form but by mitosis in the bloodstream form. Polo-like kinases (PLKs), known to play essential roles in regulating both mitosis and cytokinesis among eukaryotes, have a homologue in T. brucei, TbPLK, which regulates only cytokinesis. In our previous study, overexpressed triply hemagglutinin-tagged TbPLK (TbPLK-3HA) in the procyclic form localized to a mid-dorsal point and the anterior tip of the cell along the flagellum attachment zone (FAZ). In our current study, TbPLK-3HA expressed at the endogenous level was identified at the same dorsal location of both procyclic and bloodstream forms, albeit it was no longer detectable at the anterior tip of the cell. Endogenously expressed TbPLK fused with an enhanced yellow fluorescent protein (EYFP) localized to the same dorsal location along the FAZs in living procyclic and bloodstream cells. Fluorescence-activated cell sorter analysis of hydroxyurea-synchronized procyclic cells revealed that TbPLK-EYFP emerges during S phase, persists through G(2)/M phase, and vanishes in G(1) phase. An indicated TbPLK-EYFP association with the FAZs of G(2)/M cells may thus represent a timely localization to a potential initiation site of cytokinesis, which agrees with the recognized role of TbPLK in cytokinetic initiation.

Nuclear magnetic resonance tomography of meniscus lesions

In magnetic resonance imaging (MR) the use of suitable surface coils combined with high resolution makes it possible to obtain differentiated information on the condition of the menisci. In particular, the course and extent of cracks, degenerative changes, and the attachment zones of the menisci can be assessed. This paper describes MR and clinical findings in 58 patients and compares them with those in seven healthy subjects.

Closure of Type I Endoleaks and Landing Zone Preparation of the Thoracic Aorta

Type I endoleaks occasionally persist after endovascular repair of thoracic aneurysms. A technique for repair is described using dimensions obtained from preoperative imaging, in which the aorta is banded at the endograft attachment zone. Aortic remodeling and aortic dimensional stability are the mainstays of the approach. Measured banding has also proved to be useful for the preparation of a landing zone prior to endograft placement during hybrid procedures.

A Repetitive Protein Essential for the Flagellum Attachment Zone Filament Structure and Function in Trypanosoma brucei

The flagellum is attached along the length of the cell body in the protozoan parasite Trypanosoma brucei and is a defining morphological feature of this parasite. The flagellum attachment zone (FAZ) is a complex structure and has been characterised morphologically as comprising a FAZ filament structure and the specialised microtubule quartet (MtQ) plus the specialised areas of flagellum: plasma membrane attachment. Unfortunately, we have no information as to the molecular identity of the FAZ filament components. Here, by screening an expression library with the monoclonal antibody L3B2 which identifies the FAZ filament we identify a novel repeat containing protein FAZ1. It is kinetoplastid-specific and provides the first molecular component of the FAZ filament. Knockdown of FAZ1 by RNA interference (RNAi) results in the assembly of a compromised FAZ and defects in flagellum attachment and cytokinesis in procyclic trypanosomes. The complexity of FAZ structure and assembly is revealed by the use of other monoclonal antibody markers illustrating that FAZ1 is only one protein of a complex structure. The cytokinesis defects provide further evidence for the role of an attached flagellum in cellular morphogenesis in these trypanosomes.

SPERMIOGENESIS AND SPERMATOZOON ULTRASTRUCTURE OF NICOLLA WISNIEWSKII (DIGENEA: OPECOELIDAE), AN INTESTINAL PARASITE OF BROWN TROUT SALMO TRUTTA (PISCES: TELEOSTEI)

Spermiogenesis and ultrastructure of spermatozoon of Nicolla wisniewskii (Digenea, Opecoelidae), an intestinal parasite of Salmo trutta, were studied by electron microscopy. Spermiogenesis follows the general pattern found in the Digenea. It begins with the formation of a differentiation zone, including striated rootlets associated with 2 centrioles and an intercentriolar body. The flagella undergo a rotation of greater than 90 degrees. Then, their fusion with the median cytoplasmic process is proximodistal and asynchronous. A peculiarity was observed before the fusion of flagella, i.e., the attachment zones joined as 2 pairs by an electron-dense bridge. The mature spermatozoon is characterized by 2 axonemes, cortical microtubules, a nucleus, 2 mitochondria, external ornamentation, and spinelike bodies. At the posterior end of flagella, the spermatozoon is also characterized by the presence of a central element of the axoneme and without the 9 microtubule doublets. These results were compared with those of the other digeneans and, in particular, with other species of Opecoelidae. It appears that the number of cortical microtubules and their localization in the spermatozoon may be an interesting feature of their phylogeny.

Basal Body Positioning Is Controlled by Flagellum Formation in Trypanosoma brucei

To perform their multiple functions, cilia and flagella are precisely positioned at the cell surface by mechanisms that remain poorly understood. The protist Trypanosoma brucei possesses a single flagellum that adheres to the cell body where a specific cytoskeletal structure is localised, the flagellum attachment zone (FAZ). Trypanosomes build a new flagellum whose distal tip is connected to the side of the old flagellum by a discrete structure, the flagella connector. During this process, the basal body of the new flagellum migrates towards the posterior end of the cell. We show that separate inhibition of flagellum assembly, base-to-tip motility or flagella connection leads to reduced basal body migration, demonstrating that the flagellum contributes to its own positioning. We propose a model where pressure applied by movements of the growing new flagellum on the flagella connector leads to a reacting force that in turn contributes to migration of the basal body at the proximal end of the flagellum.

Spermiogenesis and sperm ultrastructure of Deropristis Inflata Molin, 1859 (Digenea, Deropristidae), a parasite of Anguilla anguilla

To our knowledge, this paper describes the first ultrastructural results on spermiogenesis and the spermatozoon of a Digenea belonging to the family Deropristidae, Deropristis inflata. Spermiogenesis follows the usual pattern found in the digeneans, but a single noteworthy characteristic concerns the centriole, which presents a "prominent pear-shaped electron-dense region." Spermiogenesis in D. inflata begins with the formation of a differentiation zone. The two centrioles give rise to flagella. These two flagella undergo a rotation of 90 degrees and fuse with the median cytoplasmic process. The proximo-distal fusion occurs at the level of attachment zones. The mature spermatozoon of D. inflata possesses five regions and presents all the features found in the digenean gamete. However, several characteristics allow us to distinguish the spermatozoon of D. inflata from other digenetic trematoda, namely the external ornamentations of the plasmic membrane, the anterior and posterior extremities. Furthermore, the distal part of the nucleus in front of those of the mitochondria appears such a distinctive criterion in the spermatozoon of this species. Our ultrastructural investigations produce new data on the reproduction of D. inflata, which may be useful for the understanding of the phylogenetic relationships in the Deropristidae family.

Measurement of the mechanical properties of isolated tectorial membrane using atomic force microscopy

The tectorial membrane (TM) is an extracellular matrix situated over the sensory cells of the cochlea. Its strategic location, together with the results of recent TM-specific mutation studies, suggests that it has an important role in the mechanism by which the cochlea transduces mechanical energy into neural excitation. A detailed characterization of TM mechanical properties is fundamental to understanding its role in cochlear mechanics. In this work, the mechanical properties of the TM are characterized in the radial and longitudinal directions using nano- and microindentation experiments conducted by using atomic force spectroscopy. We find that the stiffness in the main body region and in the spiral limbus attachment zone does not change significantly along the length of the cochlea. The main body of the TM is the softest region, whereas the spiral limbus attachment zone is stiffer, with the two areas having averaged Young's modulus values of 37 +/- 3 and 135 +/- 14 kPa, respectively. By contrast, we find that the stiffness of the TM in the region above the outer hair cells (OHCs) increases by one order of magnitude in the longitudinal direction, from 24 +/- 4 kPa in the apical region to 210 +/- 15 kPa at the basilar end of the TM. Scanning electron microscopy analysis shows differences in the collagen fiber arrangements in the OHC zone of the TM that correspond to the observed variations in mechanical properties. The longitudinal increase in TM stiffness is similar to that found for the OHC stereocilia, which supports the existence of mechanical coupling between these two structures.

SPERMIOGENESIS AND SPERM ULTRASTRUCTURE OF CYATHOCEPHALUS TRUNCATUS (PALLAS, 1781) KESSLER, 1868 (CESTODA: SPATHEBOTHRIIDEA)

Spermiogenesis and sperm ultrastructure of adult Cyathocephalus truncatus, a member of presumably basal group of "true" cestodes (Eucestoda), have been examined for the first time by using transmission electron microscopy. The process of sperm formation corresponds in basic pattern to that of the Pseudophyllidea. In addition, the 2 pairs of electron-dense attachment zones are present in median cytoplasmic process of C. truncatus. However, mature spermatozoa of C. truncatus differ significantly from those of the pseudophyllideans, especially in the morphology of the proximal and distal spermatozoon extremities. The proximal extremity of the mature spermatozoon lacks a crested body, which is present in more derived cestodes and some pseudophyllideans. The distal end of the mature spermatozoa exhibits different morphology in the gametes from testes and those from receptaculum seminis. New for the Eucestoda is a finding that a lateral cytoplasmic extension creates the distal end of the spermatozoa from testes, resembling sperm of some Monogenea and Digenea. In contrast, the distal extremity of the spermatozoa from receptaculum seminis contains only a nucleus. Despite the above-mentioned peculiarities, the ultrastructural data on sperm/spermiogenesis suggest close relationships of the Spathebothriidea and Pseudophyllidea.

The immunohistochemical expression of CD34 in human hair follicles: a comparative study with the bulge marker CK15

Anti-CD34 antibodies label the bulge region of mouse hair follicles. However, in human hair follicles, CD34 immunoreactivity is found in the outer root sheath below the bulge zone. The immunohistochemical staining of CD34 in catagen and telogen follicles has not been evaluated. To characterize the expression of CD34 immunoreactivity at different stages of the hair cycle in human terminal hair follicles, and to compare the immunostaining pattern of CD34 with that of CK15, used here as a marker of the bulge region. Serial vertical sections of human hair follicles in anagen, catagen and telogen phases were immunostained with anti-CD34 (QBEnd 10) and anti-CK15 (LHK15 and C8/144B) antibodies. Double-labelling immunofluorescence was also performed. The catagen and telogen follicles studied did not show CD34 immunoreactivity in the outer root sheath. The location of CD34 and CK15 immunoreactivity in anagen follicles reveals a different staining pattern: CD34-positive cells are located in the outer root sheath below the attachment zone of the arrector pili muscle, whereas CK15-positive cells are located in the outer root sheath above the attachment zone of the arrector pili muscle. Only anagen human hair follicles show CD34 immunoreactivity. CD34 and CK15 recognize different types of cells or cells at different stages of differentiation.

Trypanin, a Component of the Flagellar Dynein Regulatory Complex, Is Essential in Bloodstream Form African Trypanosomes

The Trypanosoma brucei flagellum is a multifunctional organelle with critical roles in motility, cellular morphogenesis, and cell division. Although motility is thought to be important throughout the trypanosome lifecycle, most studies of flagellum structure and function have been restricted to the procyclic lifecycle stage, and our knowledge of the bloodstream form flagellum is limited. We have previously shown that trypanin functions as part of a flagellar dynein regulatory system that transmits regulatory signals from the central pair apparatus and radial spokes to axonemal dyneins. Here we investigate the requirement for this dynein regulatory system in bloodstream form trypanosomes. We demonstrate that trypanin is localized to the flagellum of bloodstream form trypanosomes, in a pattern identical to that seen in procyclic cells. Surprisingly, trypanin RNA interference is lethal in the bloodstream form. These knockdown mutants fail to initiate cytokinesis, but undergo multiple rounds of organelle replication, accumulating multiple flagella, nuclei, kinetoplasts, mitochondria, and flagellum attachment zone structures. These findings suggest that normal flagellar beat is essential in bloodstream form trypanosomes and underscore the emerging concept that there is a dichotomy between trypanosome lifecycle stages with respect to factors that contribute to cell division and cell morphogenesis. This is the first time that a defined dynein regulatory complex has been shown to be essential in any organism and implicates the dynein regulatory complex and other enzymatic regulators of flagellar motility as candidate drug targets for the treatment of African sleeping sickness.

Serum-Stable RNA Aptamers to an Invariant Surface Domain of Live African Trypanosomes

African trypanosomes are extracellular blood parasites that cause sleeping sickness in humans and Nagana in cattle. The therapeutics used to control and treat these diseases are very ineffective and thus, the development of new drugs is urgently needed. We have previously suggested to use trypanosome-specific RNA aptamers as tools for the development of novel trypanocidal compounds. Here, we report the selection of a 2'-NH(2)-modified RNA aptamer that binds to live trypanosomes with an affinity of 70 +/- 15 nM. The aptamer adopts a stable G-quartet structure and has a half-life in human serum of > 30 h. RNA binding is restricted to the flagellar attachment zone, located between the cell body and the flagellum of the parasite. We demonstrate that antigen-tagged preparations of the aptamer can bind to live trypanosomes and that they can be used to re-direct immunoglobulins to the parasite surface.

The ruffled border and attachment regions of the apposing membrane of resorbing osteoclasts as visualized from the cytoplasmic face of the membrane

The aim of our present research was to visualize how the plasma membrane is modified and how the cytoskeleton interacts with the attachment and ruffled border regions of resorbing osteoclasts. In order to view the surface modification of membranes and associated cytoskeleton, we employed the method of cell-shearing combined with quick-freezing and rotary replication to expose and replicate an extensive area of the cytoplasmic face of the surface membrane of osteoclasts in contact with synthetic apatite as a substratum. The membrane apposed to the apatite was composed of three different domains: the attachment zone, ruffled border and the remainder. In the attachment zone, a highly organized actin filament network formed dot-shaped, F-actin rich adhesion sites, so-called podosomes, and the actin ring. The cytoskeletal filament of podosomes and actin ring appeared to be in direct contact with the cytoplasmic surface of the underlying membrane. Within the actin ring, individually recognizable podosomes were well preserved, which indicates that the actin ring was probably derived from the fusion of podosomes. After shearing at the ruffled border region, the ruffled border projections and membrane regions among the projections were left behind. These ruffled border projections contained the cytoskeletal network. These actin networks also appeared to be in direct contact with the inner side of the ruffled border membrane or in contact with it via membrane-associated particles. At the basal portion of the ruffled border, numerous clathrin-coated patches or pits were well preserved. Deeper clathrin-coated pits and vesicles were also found, which indicates an active site for receptor-mediated endocytotic events. Clathrin sheets were also observed in the cell periphery outside of the actin ring. This type of clathrin sheets adhered to the apatite substrate, but was not anchored to the actin microfilaments. Our study thus clearly visualized the interaction between the cytoskeletal filaments and the underlying membrane at the ruffled border, attachment zone and podosome in osteoclasts cultured on apatitepellets.

The geometric pattern of a pillared substrate influences the cell-process distribution and shapes of fibroblasts

Fibroblasts alter their shape, direction of movement, cytoskeleton arrangement, and focal contact when placed upon square array pillars. We prepared pillars of 1 μm diameter, separated by 3 μm, and having 1, 5, and 10 μm heights using substrates displaying identical surface chemistry. When cells seeded initially onto the tops of the pillars, fibroblasts subsequently were immobilized in situ by several pillars that visibly protruded through, but did not pierce, the cell bodies. The cytoplasma then migrated outward with long straight lamella along the interval of the pillars and formed several discrete attachment zones at their side walls – the value of their form index (FI) was as high as 35 – which altered the cellular shape entirely. Most of the cells interacted with the pillar substrate by spreading preferentially in a particular direction, but some of them had the ability to undergo coincident two-direction ( x and y) migration; right-angle turn orientations led to the growth of dramatic cellular morphologies. Interestingly, this fibroblast's behavior variation was gradually in proportion to the pillar height of substrate. Our results confirm that cellular migration and cellular shape are both strongly affected by the geometry of the growth microenvironment.

Regulation of Integrin Activity by MIA

MIA (melanoma inhibitory activity) has been identified as a small protein secreted from malignant melanoma cells, which interacts with extracellular matrix proteins including fibronectin. Here, we show that MIA negatively regulates the activity of the mitogen-activated protein kinase pathway in malignant melanoma. Using far Western blotting and co-immunoprecipitation we searched for MIA-binding cell surface proteins. We found that MIA interacts with integrin alpha4beta1 and alpha5beta1, leading to down-regulation of integrin activity and reduction of mitogen-activated protein kinase signaling. These findings also suggest that MIA may play a role in tumor progression and the spread of malignant melanomas via mediating detachment of cells from extracellular matrix molecules by modulating integrin activity. Inhibiting MIA functions in vivo may therefore provide a novel therapeutic strategy for metastatic melanoma disease.

Spermatological characters of the pseudophyllidean cestode Bothriocephalus scorpii (Müller, 1776)

Spermiogenesis of Bothriocephalus scorpii (Cestoda, Pseudophyllidea) includes an orthogonal development of two flagella, followed by a flagellar rotation and a proximo-distal fusion with the median cytoplasmic process. The fusion occurs at the level of four attachment zones. The presence of dense material in the apical region of the differentiation zone in the early stage of spermiogenesis appears to be a characteristic feature for the Pseudophyllidea. The mature spermatozoon possesses two axonemes of 9+“1” pattern of the Trepaxonemata, nucleus, cortical microtubules, electron-dense granules and crested body. The anterior part of the gamete exhibits a centriole surrounded by electron-dense tubular structures arranged as incomplete spiral. When the crested body disappears, the electron-dense tubular structures are arranged into a ring encircling the axoneme. The electron-dense tubular structures and their arrangement appear to be a specific feature for the clade “Bothriocephalidea”. The organization of the posterior extremity of the gamete with the nucleus is described for the first time in the Pseudophyllidea.

Spermatological characters inDiphyllobothrium latum (Cestoda, Pseudophyllidea)

Spermiogenesis and the ultrastructural features of the spermatozoon of Diphyllobothrium latum (Cestoda, Pseudophyllidea) are described using transmission electron microscopy. Spermiogenesis is characterized by the development of two flagella of unequal length that grow asynchronously. When the first growing flagellum starts to rotate, the second one develops. Flagellar rotation is thus asymmetric and asynchronic. It is followed by proximodistal fusion with the median cytoplasmic process. Electron-dense material is present in the apical region of the zone of differentiation in the early stages of spermiogenesis. The intercentriolar body consists of seven plates: three are electron-dense. Four attachment zones occur in the median cytoplasmic process. An atypical arrangement of striated roots was occasionally observed. The mature spermatozoon possesses two axonemes of 9 + "1" trepaxonematan pattern, nucleus, cortical microtubules, electron-dense granules, and lacks mitochondria. The ultrastructure of the anterior extremity of the spermatozoon in D. latum clearly differs from that in the bothriocephalid pseudophyllideans, mainly in the absence of a crested body and a ring of electron-dense tubular structures. The spermatological data support the assumption that the order Pseudophyllidea is formed by two unrelated clades, "Bothriocephalidea" and "Diphyllobothriidea."

Dissociation of Cytokinesis Initiation from Mitotic Control in a Eukaryote

Cytokinesis is initiated only after mitotic exit in eukaryotes. However, in the insect (procyclic) form of an ancient protist, Trypanosoma brucei, a blockade at the G2/M checkpoint results in an enrichment of anucleate cells (zoids), suggesting separated regulations between mitosis and cytokinesis (X. Tu and C. C. Wang, J. Biol. Chem. 279:20519-20528, 2004). Polo-like kinases (Plks) are known to play critical roles in controlling both mitosis and cytokinesis. A single Plk homologue in T. brucei, TbPLK, was found to be capable of complementing the Plk (Cdc5) functions in Saccharomyces cerevisiae, thus raising the question of how it may function in the trypanosome with cytokinesis dissociated from mitosis. Depletion of TbPLK in the procyclic form of T. brucei by RNA interference resulted in growth arrest with accumulation of multiple nuclei, kinetoplasts, basal bodies, and flagella in approximately equal numbers among individual cells. There were, however, few zoids detectable, indicating inhibited cytokinesis with unblocked mitosis and kinetoplast segregation. TbPLK is thus apparently involved only in initiating cytokinesis in T. brucei. Overexpression of TbPLK in the trypanosome did not affect cell growth, but 13% of the resulting population was in the zoid form, suggesting runaway cytokinesis. An immunofluorescence assay indicated that TbPLK was localized in a chain of likely flagellum attachment zones in the cytoskeleton. In a dividing cell, a new line of such zones appeared closely paralleling the existing one, which could constitute the cleavage furrow. An exposed region of TbPLK at the anterior tip of the cell may provide the trigger of cytokinesis. Taken together, our results revealed a novel mechanism of cytokinesis initiation in the trypanosome that may serve as a useful model for further in-depth investigations.

The flagellar attachment zone of Trypanosoma cruzi epimastigote forms

The flagellar attachment zone (FAZ) is an adhesion region of Trypanosoma cruzi epimastigote forms where the flagellum emerges from the flagellar pocket and remains attached to the cell body. This region shows a junctional complex which is formed by a linear series of apposed macular structures that are separated by amorphous material and clusters of intramembranous particles. Two protein groups appear to be important in the FAZ region: a membrane glycoprotein of 72 kDa and several high molecular weight proteins. To gain a better understanding of the FAZ region, we compared wild-type Y strain T. cruzi epimastigotes with a mutant cell in which the 72-kDa surface glycoprotein (Gp72), involved in cell body-flagellum adhesion, had been deleted by target gene replacement. Using immunofluorescence confocal microscopy and electron microscopy techniques to analyze the FAZ region the results suggest that, in the absence of Gp72, other proteins involved in the formation of FAZ remain concentrated in the flagellar pocket region. The analysis of a 3-D reconstruction model of wild-type epimastigotes showed that the endoplasmic reticulum and mitochondrion are in intimate association with FAZ, in contrast to the null mutant cells where the endoplasmic reticulum was not visualized.

Novel Extracellular Vesicles Mediate an ABCG2-Dependent Anticancer Drug Sequestration and Resistance

Overexpression of the multidrug efflux transporter ABCG2 in the plasma membrane of cancer cells confers resistance to various anticancer drugs, including mitoxantrone. Here, we explored the mechanism underlying drug resistance in the MCF-7 breast cancer sublines MCF-7/MR and MCF-7/FLV1000 cells in which wild-type (R482) ABCG2 overexpression is highly confined to cell-cell attachment zones. The latter comprised the membrane of novel extracellular vesicles in which mitoxantrone was rapidly and dramatically sequestered. After 12 hours of incubation with mitoxantrone, the estimated intravesicular drug concentration was approximately 1,000-fold higher than in the culture medium. This drug compartmentalization was prevented by the specific and potent ABCG2 transport inhibitors Ko143 and fumitremorgin C, thereby resulting in restoration of drug sensitivity. Consistently, this intravesicular drug concentration was abrogated by energy deprivation and was restored upon provision of energy substrates. Fine-structure studies corroborated the presence of numerous large extracellular vesicles that were highly confined to cell-cell attachment zones between neighbor cells. Furthermore, high-resolution electron microscopy revealed that the membrane of these extracellular vesicles contained microvilli-like invaginations protruding into the intravesicular lumen. It is likely that these microvilli-like projections increase the vesicular membrane surface, thereby allowing for a more efficient ABCG2-dependent intravesicular anticancer drug concentration. Hence, these novel extracellular vesicles mediate the ABCG2-dependent extraction of intracellular drug, thereby serving as cytotoxic drug disposal chambers shared by multiple neighbor cancer cells. This constitutes a novel modality of anticancer drug resistance.