C-Terminal Tail Polyglycylation and Polyglutamylation Alter Microtubule Mechanical Properties.

Microtubules are biopolymers that perform diverse cellular functions. Microtubule behavior regulation occurs in part through post-translational modification of both the α- and ß-subunits of tubulin. One class of modifications is the heterogeneous addition of glycine and/or glutamate residues to the disordered C-terminal tails (CTTs) of tubulin. Because of their prevalence in stable, high-stress cellular structures such as cilia, we sought to determine if these modifications alter microtubules' intrinsic stiffness. Here, we describe the purification and characterization of differentially modified pools of tubulin from Tetrahymena thermophila. We found that post-translational modifications do affect microtubule stiffness but do not affect the number of protofilaments incorporated into microtubules. We measured the spin dynamics of nuclei in the CTT backbone by NMR spectroscopy to explore the mechanism of this change. Our results show that the α-tubulin CTT does not protrude out from the microtubule surface, as is commonly depicted in models, but instead interacts with the dimer's surface. This suggests that the interactions of the α-tubulin CTT with the tubulin body contributes to the stiffness of the assembled microtubule, thus providing insight into the mechanism by which polyglycylation and polyglutamylation can alter microtubule mechanical properties.

Mechanisms of microtubule dynamics and force generation examined with computational modeling and electron cryotomography.

Microtubules are dynamic tubulin polymers responsible for many cellular processes, including the capture and segregation of chromosomes during mitosis. In contrast to textbook models of tubulin self-assembly, we have recently demonstrated that microtubules elongate by addition of bent guanosine triphosphate tubulin to the tips of curving protofilaments. Here we explore this mechanism of microtubule growth using Brownian dynamics modeling and electron cryotomography. The previously described flaring shapes of growing microtubule tips are remarkably consistent under various assembly conditions, including different tubulin concentrations, the presence or absence of a polymerization catalyst or tubulin-binding drugs. Simulations indicate that development of substantial forces during microtubule growth and shortening requires a high activation energy barrier in lateral tubulin-tubulin interactions. Modeling offers a mechanism to explain kinetochore coupling to growing microtubule tips under assisting force, and it predicts a load-dependent acceleration of microtubule assembly, providing a role for the flared morphology of growing microtubule ends.

Ultrastructural Analysis of Microtubule Ends.

Microtubules can be detected in light microscopes, but the limited resolution of these instruments means that the polymers appear as lines whose width is defined by the diffraction of light. Much important work on microtubule dynamics has been accomplished by light microscopy, but the details of microtubule end structure are not accessible in such studies. Slight variations in fluorescence intensity, etc. have been used to comment on the structure of dynamic ends, and the combination of light microscopy with laser tweezers has provided insight into aspects of microtubule elongation. However, for views that reveal structural details of the pathways for microtubule growth and shortening, electron microscopy has been of great value. Here, we describe methods for using electron microscopes to look at the ends of microtubules as they grow and shrink, both in vivo and in vitro. The key problems to be overcome for ultrastructural study of microtubule dynamics are those of reliable sample preparation. Dynamic microtubules are labile and can therefore be modified by preparative methods. Our chapter follows the premise that rapid freezing, which converts sample water into vitreous ice, is the best approach for sample preparation. Therefore, all of the methods described involve finding optimal conditions for sample vitrification, and then getting the frozen sample into a form suitable for electron microscopy. We also posit that the end of a microtubule must be considered in three dimensions, so we employ electron tomography as a way to get the necessary information. The methods described for the study of microtubules in cells employ rapid freezing, freeze-substitution fixation, plastic embedding, serial sectioning, and tomography of stained samples. The methods for following microtubule growth in vitro employ sample preparation on holy grids, blotting, and plunge-freezing, followed by electron cryo-tomography. Quantification of structure from both approaches is accomplished by segmentation and analysis of graphic models.

A Simple and Effective Method for Flow Cytometric Study of Lymphoid Malignancies Using Needle Core Biopsy Specimens.

OBJECTIVES: We developed a simple and effective rinsing technique (RT) of needle biopsies to produce cell suspensions for flow cytometry (FCM) and evaluated whether the RT is comparable to the conventional tissue cell suspension (TCS) technique. METHODS: We retrieved 93 needle core biopsy cases employing the RT for FCM and 25 needle biopsy cases using TCS for FCM. RESULTS: The diagnostic concordance between the FCM results and the morphologic diagnoses of both groups was compared. The diagnostic concordance was comparable in the RT group (92.6%) to the TCS group (71.4%). Furthermore, the diagnostic concordance in the RT group was associated with number of isolated cells. The diagnostic accuracy increased significantly when the cell number was above 30,000 in the RT group. CONCLUSIONS: The RT for FCM not only maximizes the tissue utilization, but also is a simple and effective method to obtain cell suspension as compared to traditional cell suspension technique. © 2018 International Clinical Cytometry Society.

Multiple dynamin family members collaborate to drive mitochondrial division.

Mitochondria cannot be generated de novo; they must grow, replicate their genome, and divide in order to be inherited by each daughter cell during mitosis. Mitochondrial division is a structural challenge that requires the substantial remodelling of membrane morphology. Although division factors differ across organisms, the need for multiple constriction steps and a dynamin-related protein (Drp1, Dnm1 in yeast) has been conserved. In mammalian cells, mitochondrial division has been shown to proceed with at least two sequential constriction steps: the endoplasmic reticulum and actin must first collaborate to generate constrictions suitable for Drp1 assembly on the mitochondrial outer membrane; Drp1 then further constricts membranes until mitochondrial fission occurs. In vitro experiments, however, indicate that Drp1 does not have the dynamic range to complete membrane fission. In contrast to Drp1, the neuron-specific classical dynamin dynamin-1 (Dyn1) has been shown to assemble on narrower lipid profiles and facilitate spontaneous membrane fission upon GTP hydrolysis. Here we report that the ubiquitously expressed classical dynamin-2 (Dyn2) is a fundamental component of the mitochondrial division machinery. A combination of live-cell and electron microscopy in three different mammalian cell lines reveals that Dyn2 works in concert with Drp1 to orchestrate sequential constriction events that build up to division. Our work underscores the biophysical limitations of Drp1 and positions Dyn2, which has intrinsic membrane fission properties, at the final step of mitochondrial division.

Three-dimensional cryo-electron microscopy on intermediate filaments.

Together with microtubules and actin filaments (F-actin), intermediate filaments (IFs) form the cytoskeleton of metazoan cells. However, unlike the other two entities that are extremely conserved, IFs are much more diverse and are grouped into five different families. In contrast to microtubules and F-actin, IFs do not exhibit a polarity, which may be the reason that no molecular motors travel along them. The molecular structure of IFs is less well resolved than that of the other cytoskeletal systems. This is partially due to their functional variability, tissue-specific expression, and their intrinsic structural properties. IFs are composed mostly of relatively smooth protofibrils formed by antiparallel arranged α-helical coiled-coil bundles flanked by small globular domains at either end. These features make them difficult to study by various electron microscopy methods or atomic force microscopy (AFM). Furthermore, the elongated shape of monomeric or dimeric IF units interferes with the formation of highly ordered three-dimensional (3-D) crystals suitable for atomic resolution crystallography. So far, most of the data we currently have on IF macromolecular structures come from electron microscopy of negatively stained samples, and fragmented α-helical coiled-coil units solved by X-ray diffraction. In addition, AFM allows the observation of the dynamic states of IFs in solution and delivers a new view into the assembly properties of IFs. Here, we discuss the applicability of cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) for the field. Both methods are strongly related and have only recently been applied to IFs. However, cryo-EM revealed distinct new features within IFs that have not been seen before, and cryo-ET adds a 3-D view of IFs revealing the path and number of protofilaments within the various IF assemblies.

Mantle cell lymphoma with flow cytometric evidence of clonal plasmacytic differentiation: a case report.

BACKGROUND: Plasmacytic differentiation in mantle cell lymphoma (MCL) occurs rarely. However, no flow cytometric studies that demonstrate plasmacytic (PC) differentiation in MCL have been reported. Herein, we report a case of MCL with PC differentiation identified by flow cytometry. METHODS: Morphologic review was performed by hematoxylin and eosin (H&E) stained sections from paraffin-embedded lymph node, colon and bone marrow specimens, and Wright-Geimsa stained bone marrow aspirate smears and touch imprints. Immunohistochemical stains using antibodies against CD3, CD5, CD20, and cyclin-D1, and in-situ hybridization for kappa and lambda light chains were reviewed. Multicolor flow cytometry analysis was performed on the bone marrow aspirate with monoclonal antibodies to CD3, CD4, CD5, CD8, CD14, CD19, CD20, CD23, CD38, CD45, CD56, CD138, and kappa and lambda light chains. FISH analysis for t(11;14)(q13;q32) was performed on interphase cells. RESULTS: The neoplastic cells had the cytologic features of MCL with nodal, bone marrow, and colonic involvement. In-situ hybridization for kappa and lambda light chains demonstrated clonal plasma cells in the lymph node and bone marrow biopsies. In addition, flow cytometric studies of the bone marrow aspirate showed three populations of neoplastic cells: a clonal B-cell population with typical MCL phenotype, a similar B-cell population in transition to plasma cells, and a clonal plasma cell population. The plasma cells retained CD5 expression and had the same light chain restriction as the clonal B-cells. CONCLUSIONS: Multi-parameter flow cytometry can be useful in demonstrating clonal PC differentiation in MCL and distinguishing from a concurrent but unrelated plasma cell dyscrasia.

Familial B-cell chronic lymphocytic leukemia: analysis of cytogenetic abnormalities, immunophenotypic profiles, and immunoglobulin heavy chain gene usage.

B-cell chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease that may exhibit familial clustering. We examined the cytogenetic, immunophenotypic, and VH gene usage characteristics of a family with B-CLL affecting 7 members in 3 generations. Interphase fluorescence in situ hybridization studies identified an acquired deletion of chromosome 13q14 in the leukemic cells of 6 affected members, accompanied by deletion 14q32 or trisomy 12 in 2 cases. VH gene analysis demonstrated clonal rearrangements of the VH3 gene family in 5 cases and of VH2 genes in 1 case. All 6 cases were mutated in VH2 or VH3. Two cases had a second VH1 family gene rearrangement that was unmutated. Flow cytometry performed on 5 cases showed the typical B-CLL immunophenotype; all were CD38-, but 3 expressed ZAP-70. Our findings support previous observations that familial and sporadic B-CLL cases are biologically similar and suggest that familial clusters will be useful for studying pathogenetic events in B-CLL.