Rapid Visualization of Microtubules in Blood Cells and Other Cell Types in Marine Model Organisms

Kg, Lee; Braun, Armin; Chaikhoutdinov, Irina; J, DeNobile; Conrad, Mara; Cohen, William

doi:10.2307/1543398

Cited by 8 publications

(4 citation statements)

References 3 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Microtubule-based marginal bands confer ellipsoid shapes on certain nucleated invertebrate hematocytes and vertebrate blood cells 45 including blood platelets. Classic electron microscopy studies by Behnke and Zelander, 11,12 White, 13 Nachmias, 14 and Kenney and Linck 3 provided insight into the structure of the microtubule ring in platelets and …”

Section: Discussionmentioning

confidence: 99%

Visualization of microtubule growth in living platelets reveals a dynamic marginal band with multiple microtubules

Patel–Hett

Richardson

Schulze

et al. 2008

Blood

139

140

View full text Add to dashboard Cite

The marginal band of microtubules maintains the discoid shape of resting blood platelets. Although studies of platelet microtubule coil structure conclude that it is composed of a single microtubule, no investigations of its dynamics exist. In contrast to previous studies, permeabilized platelets incubated with GTPrhodamine-tubulin revealed tubulin incorporation at 7.9 (؎ 1.9) points throughout the coil, and anti-EB1 antibodies stained 8.7 (؎ 2.0) sites, indicative of multiple free microtubules. To pursue this result, we expressed the microtubule plus-end marker EB3-GFP in megakaryocytes and examined its behavior in living platelets released from these cells. Time-lapse microscopy of EB3-GFP in resting platelets revealed multiple assembly sites within the coil and a bidirectional pattern of assembly. Consistent with these findings, tyrosinated tubulin, a marker of newly assembled microtubules, localized to resting platelet microtubule coils. These results suggest that the resting platelet marginal band contains multiple highly dynamic microtubules of mixed polarity. Analysis of microtubule coil diameters in newly formed resting platelets indicates that microtubule coil shrinkage occurs with aging. In addition, activated EB3-GFP-expressing platelets exhibited a dramatic increase in polymerizing microtubules, which travel outward and into filopodia. Thus, the dynamic microtubules associated with the marginal band likely function during both resting and activated platelet states. IntroductionPlatelets are cells that function in maintaining vascular integrity. Their disclike shape enables platelets to travel along the apical endothelium of vessels, where they respond to vascular damage by activating and releasing hemostatic factors. Situated beneath the plasma membrane is a circumferential marginal band composed of 7 to 12 filamentous rings that maintain the discoid shape of resting platelets. Marginal bands assemble in blood cells of other species, but platelets are the only mature human cell possessing a circumferential marginal band. 1 The platelet marginal band is composed almost entirely of microtubules. 2,3 Microtubules are polymers of ␣␤-tubulin dimers that first associate into linear arrays called protofilaments. Protofilaments laterally associate forming the hollow rigid tubular structure characteristic of microtubules. ␤1-Tubulin, a divergent ␤-tubulin isoform exclusive to megakaryocytes and platelets, 4,5 makes up the bulk of ␤-tubulin within the microtubule coil. 3,6 The marginal band preserves the elliptic shape of resting platelets. Transgenic mice lacking ␤1-tubulin possess nondiscoid platelets with defective marginal bands containing only 2 to 3 microtubule coils. 7,8 ␤1-Tubulin-deficient mice experience thrombocytopenia (platelet counts Ͻ 50% of wild-type) and prolonged bleeding times. 8 Chilling of platelets from wild-type mice disassembles their microtubules and induces spherocytosis (spherical shape). Stabilization of the microtubule coil with paclitaxel prior to chilling prevents spherocytosis ...

show abstract

Section: Discussionmentioning

confidence: 99%

Visualization of microtubule growth in living platelets reveals a dynamic marginal band with multiple microtubules

Patel–Hett

Richardson

Schulze

et al. 2008

Blood

139

140

View full text Add to dashboard Cite

show abstract

“…Morphologically, the two cell types are quite distinct: unactivated platelets are highly flattened, minute (w3 µm diameter), anucleate discoids, whereas fish, amphibian, reptilian, and avian thrombocytes are thicker nucleated ellipsoids of more typical eucaryotic size (w10 µm or greater on the long axis; Mainwaring and Rowley, 1985;Shepro et al, 1966). Despite these differences in size and shape, unactivated non-mammalian thrombocytes, referred to hereafter as nucleated thrombocytes, have two highly distinctive structural features in common with platelets: a "canalicular" membrane system just beneath their surface (Daimon and Uchida, 1985;White and Clawson, 1980), and a prominent marginal band of microtubules (MB) in their plane of flattening (Fawcett and Witebsky, 1964;Lee et al, 2002;Rowley et al, 1988;Schwer et al, 2001). Upon activation of both nucleated thrombocytes and platelets, cellular aggregation, adhesion and morphological alterations are observed.…”

Section: Introductionmentioning

confidence: 99%

Shape transformation and cytoskeletal reorganization in activated non‐mammalian thrombocytes

Lee

Miller

Anastassov

et al. 2004

Cell Biology International

View full text Add to dashboard Cite

The nucleated thrombocytes of non-mammalian vertebrates are partially flattened, ovoid cells morphologically distinct from mammalian platelets, and the extent of their functional equivalence is unknown. To test whether they resemble platelets in having similar F-actin-based post-activation stages, rapid fixation/extraction/labeling methods were developed to reveal cytoskeletal organization in dogfish thrombocytes by confocal microscopy. Unactivated cells contained cortical F-actin plus denser F-actin co-localizing with outer marginal band (MB) microtubules. In the post-activation sequence, determined for the first time by continuous observation of individual thrombocytes following thrombin perfusion, cells rounded and blebbed, spread, and eventually flattened extensively. The MB twisted and then became disorganized, with microtubule bundles remaining centrally located and associated with nuclear clefts. In contrast, F-actin occupied blebs and outward-spreading cytoplasm, initially in spiky projections, then predominantly in stress fibers, and inhibitors of F-actin assembly or myosin ATPase blocked shape changes. Thus, the post-activation stages and cytoskeletal events observed in nucleated thrombocytes were found to parallel those of platelets.

show abstract

“…Methods for in situ hybridization and immunofluorescence were similar to those described in an earlier report (20) with an important modification. In the present study, oocytes were fixed for 1 h at room temperature in 4% formaldehyde containing 0.6% Brij 58, 5 mM EGTA, and 1 mM MgCl 2 , buffered with 100-mM pipes, pH 6.8 (27). They were then rinsed once briefly in PBS with 0.1% Tween-20 (PBST), equilibrated in PBST for 30 min, dehydrated in a series of ethanols, and stored at −20°C in 70% ethanol.…”

Section: Methodsmentioning

confidence: 99%

Rediscovery of the nucleolinus, a dynamic RNA-rich organelle associated with the nucleolus, spindle, and centrosomes

Alliegro¹,

Henry

Alliegro³

2010

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The nucleolinus is an RNA-rich compartment, closely apposed to or embedded within the nucleolus. Discovered over 150 y ago, fewer than two dozen articles have been published on the nucleolinus, probably because complex histochemical stains are required for its visualization in the great majority of cells. The nucleolinus has been reported in invertebrate oocytes, mammalian and amphibian epithelial cells, neurons, and several transformed cell lines. A prominent nucleolinus, clearly visible with transmitted light microscopes at 10× magnification, is present in each oocyte of the surf clam, Spisula solidissima. We observed a consistent relationship between the nucleolinus and the developing meiotic apparatus following Spisula oocyte activation. Through sonication and sucrose gradient fractionation of purified oocyte nuclei, we isolated nucleolini, extracted their RNA, and prepared an in situ riboprobe (NLi-1), which is associated specifically with the nucleolinus, confirming its unique composition. Other in situ observations revealed a NLi-1 and nucleolinar association with the developing spindle and centrosomes. Laser microsurgery that targeted the nucleolinus resulted in failed meiotic cell division in parthenogenetically activated oocytes and failed mitosis in fertilized oocytes. Although the nucleolinus may be a forgotten organelle, its demonstrated role in spindle formation suggests it deserves renewed attention.cell division | mitosis | meiosis T he nucleolinus was described by Montgomery in eggs of the nudibranch mollusk, Montagua, in his 1898 monograph, "Comparative Cytological Studies, with Especial Regard to the Morphology of the Nucleolus" (1). As the title indicates, the study focuses on the appearance and behavior of the nucleolus. In it, Montgomery presents his own thoughts on the significance of the nucleolinus as well as the observations of other investigators, including Louis Agassiz, over the previous 40 y (2-4). These range from the attachment of no morphological significance to the structure to suggestions that the nucleolinus is a microorganism enclosed in the nucleus and a direct morphological progenitor of the centrosome. Twenty-two years later, in his "Observations on an Intra-Nucleolar Body," Carleton experimented with Cajal's formol-silver nitrate technique to visualize the nucleolinus in cat and frog intestinal epithelial cells (5). In some cases, he reported, the nucleolinus was the only silverimpregnated structure within the entire nucleus. As of 90 y ago, then, the nucleolinus was clearly distinguished from the surrounding or adjacent nucleolus, but only via histochemistry. Carleton, on the basis of morphological observations of fixed, sectioned tissues, suggested that the nucleolinus divided into two during prophase. Allen (6, 7) subsequently proposed a "spindle forming role" for the nucleolinus, on the basis of his observations of live Spisula oocytes, but no photomicrographs or other data were presented to directly support this hypothesis.The most comprehensive analysis of the nucle...

show abstract

Rapid Visualization of Microtubules in Blood Cells and Other Cell Types in Marine Model Organisms

Cited by 8 publications

References 3 publications

Visualization of microtubule growth in living platelets reveals a dynamic marginal band with multiple microtubules

Visualization of microtubule growth in living platelets reveals a dynamic marginal band with multiple microtubules

Shape transformation and cytoskeletal reorganization in activated non‐mammalian thrombocytes

Rediscovery of the nucleolinus, a dynamic RNA-rich organelle associated with the nucleolus, spindle, and centrosomes

Contact Info

Product

Resources

About