Microtubules In Mammalian Cells Research Articles

Super-resolution radial fluctuations microscopy for optimal resolution and fidelity.

Fluorescence fluctuation super-resolution microscopy (FF-SRM) has emerged as a promising method for the fast, low-cost, and uncomplicated imaging of biological specimens beyond the diffraction limit. Among FF-SRM techniques, super-resolution radial fluctuation (SRRF) microscopy is a popular technique but is prone to artifacts, resulting in low fidelity, especially under conditions of high-density fluorophores. In this Letter, we developed a novel, to the best of our knowledge, combinatory computational super-resolution microscopy method, namely VeSRRF, that demonstrated superior performance in SRRF microscopy. VeSRRF combined intensity and gradient variance reweighted radial fluctuations (VRRF) and enhanced-SRRF (eSRRF) algorithms, leveraging the enhanced resolution achieved through intensity and gradient variance analysis in VRRF and the improved fidelity obtained from the radial gradient convergence transform in eSRRF. Our method was validated using microtubules in mammalian cells as a standard biological model system. Our results demonstrated that VeSRRF consistently achieved the highest resolution and exceptional fidelity compared to those obtained from other algorithms in both single-molecule localization microscopy (SMLM) and FF-SRM. Moreover, we developed the VeSRRF software package that is freely available on the open-source ImageJ/Fiji software platform to facilitate the use of VeSRRF in the broader community of biomedical researchers. VeSRRF is an exemplary method in which complementary microscopy techniques are integrated holistically, creating superior imaging performance and capabilities.

Rab1A regulates anterograde melanosome transport by recruiting kinesin-1 to melanosomes through interaction with SKIP

Melanosomes are lysosome-related organelles in melanocytes that are transported from the perinucleus to the cell periphery by coordination between bidirectional (anterograde and retrograde) microtubule-dependent transport and unidirectional actin-dependent transport. Although the molecular machineries that mediate retrograde transport and actin-dependent transport have already been identified, little is known about the anterograde transport complex on microtubules in mammalian cells. Here we discovered that small GTPase Rab1A on melanosomes recruits SKIP/PLEKHM2 as a Rab1A-specific effector and that Rab1A, SKIP, and a kinesin-1/(Kif5b+KLC2) motor form a transport complex that mediates anterograde melanosome transport in melanocytes. Interestingly, Arl8, Arf-like small GTPase that also interacts with SKIP, is specifically localized at lysosomes and regulates their anterograde transport in melanocytes. Our findings suggest that the anterograde microtubule-dependent transport of melanosomes and lysosomes are differently regulated by independent cargo receptors, i.e., Rab1A and Arl8, respectively, but that a SKIP–kinesin-1 mechanism is responsible for the transport of both.

A higher-order mathematical modeling for dynamic behavior of protein microtubule shell structures including shear deformation and small-scale effects

Microtubules in mammalian cells are cylindrical protein polymers which structurally and dynamically organize functional activities in living cells. They are important for maintaining cell structures, providing platforms for intracellular transport, and forming the spindle during mitosis, as well as other cellular processes. Various in vitro studies have shown that microtubules react to applied mechanical loading and physical environment. To investigate the mechanisms underlying such phenomena, a mathematical model based on the orthotropic higher-order shear deformation shell formulation and Hamilton’s principle is presented in this paper for dynamic behavior of microtubules. The numerical results obtained by the proposed shell model are verified by the experimental data from the literature, showing great consistency. The nonlocal elasticity theory is also utilized to describe the nano-scale effects of the microtubule structure. The wave propagation and vibration characteristics of the microtubule are examined in the presence and absence of the cytosol employing proposed formulations. The effects of different system parameters such as length, small scale parameter, and cytosol viscosity on vibrational behavior of a microtubule are elucidated. The definitions of critical length and critical viscosity are introduced and the results obtained using the higher order shell model are compared with those obtained employing a first-order shear deformation theory. This comparison shows that the small scale effects become important for higher values of the wave vector and the proposed model gives more accurate results for both small and large values of wave vectors. Moreover, it is shown that for higher circumferential wave number, the torsional wave velocity obtained by the higher-order shell model tend to be higher than the one predicted by the first-order shell model.

Probing Forces on Newly Generated Spindle Microtubule Minus-Ends

The mitotic spindle is a dynamic self-organizing machine that coordinates cell division and preserves genomic stability. The ability to focus microtubule minus-ends into poles is crucial to spindle structure and function. However, our understanding of pole-focusing forces has been limited by the challenges of labeling and imaging microtubule minus-ends in established spindles. Here, we used laser ablation to sever kinetochore-fiber microtubules in mammalian cells and probe how the cell detects and organizes newly generated microtubule minus-ends. Within a few seconds of ablation, the cell recognizes new minus-ends and begins pulling them poleward. These pole-focusing forces exist throughout metaphase and anaphase and can move chromosomes rapidly, dominating other spindle forces. Opposing forces on chromosomes from the other half-spindle are able to slow, though not stop, the pole-focusing response, as indicated by faster pole-focusing speeds in monopolar spindles and during anaphase than in metaphase bipolar spindles. Together, our data indicate that microtubule minus-end focusing forces operate broadly and rapidly and are of similar magnitude to other spindle forces. These pole-focusing forces are thus well-suited to robustly maintain spindle structural integrity despite rapid turnover of spindle components and mechanical challenges.

The murine Gcap14 gene encodes a novel microtubule binding and bundling protein

Microtubules form flexible fibers, which are utilized in cell proliferation and differentiation. Although the flexibility of microtubules was shown to be regulated by various microtubule-associated proteins, this regulation is still far from complete understanding. Here, we report a new potential regulator of microtubules in mammals. Gcap14 colocalizes with microtubules in mammalian cells transfected with Gcap14 expression vector. Association of Gcap14 with microtubules was confirmed by biochemical subcellular fractionation. Recombinant Gcap14 protein cosedimented with pure microtubules, indicating a direct binding between the two. Furthermore, recombinant Gcap14 was shown to have the ability of inducing microtubule bundling in vitro. Structured summary of protein interactionsGcap14 physically interacts with Gcap14 by anti tag coimmunoprecipitation (View Interaction: 1, 2)

Cep57, a NEDD1-binding pericentriolar material component, is essential for spindle pole integrity

Formation of a bipolar spindle is indispensable for faithful chromosome segregation and cell division. Spindle integrity is largely dependent on the centrosome and the microtubule network. Centrosome protein Cep57 can bundle microtubules in mammalian cells. Its related protein (Cep57R) in Xenopus was characterized as a stabilization factor for microtubule-kinetochore attachment. Here we show that Cep57 is a pericentriolar material (PCM) component. Its interaction with NEDD1 is necessary for the centrosome localization of Cep57. Depletion of Cep57 leads to unaligned chromosomes and a multipolar spindle, which is induced by PCM fragmentation. In the absence of Cep57, centrosome microtubule array assembly activity is weakened, and the spindle length and microtubule density decrease. As a spindle microtubule-binding protein, Cep57 is also responsible for the proper organization of the spindle microtubule and localization of spindle pole focusing proteins. Collectively, these results suggest that Cep57, as a NEDD1-binding centrosome component, could function as a spindle pole- and microtubule-stabilizing factor for establishing robust spindle architecture.

New function of the proline rich domain in dynamin-2 to negatively regulate its interaction with microtubules in mammalian cells

Microtubule reorganization is necessary for many cellular functions such as cell migration, cell polarity and cell division. Dynamin was originally identified as a microtubule-binding protein. Previous limited digestion experiment revealed that C-terminal 100-amino acids proline rich domain (PRD) of dynamin is responsible for microtubule binding in vitro. However, as obvious localization of dynamin along microtubules is only observed at the spindle midzone during mitosis but not in interphase cells, it remains unclear how dynamin interacts with microtubules in vivo. Here, we report that GFP-dynamin-2-(1–786), a truncated mutant lacking a C-terminal portion of the PRD, localized along microtubules in interphase HeLa cells. GFP-dynamin-2-wild type (WT) and GFP-dynamin-2-(1–745), a construct that was further truncated to remove the entire PRD, localized in discrete punctate structures but not along microtubules. These data suggest that the N-terminal (residues 746–786) but not the entire PRD is necessary for the interaction of dynamin-2 with microtubules in the cell and that the C-terminus of PRD (787–870) negatively regulate this interaction. Microtubules in cells expressing GFP-dynamin-2-(1–786) were stabilized against exposure to cold. These results provide a first evidence for a regulated interaction of dynamin-2 with microtubules in cultured mammalian cells.

4Pi microscopy with linear fluorescence excitation

Practical 4Pi microscopy has so far exclusively relied on multiphoton excitation of fluorescence, because the nonlinear suppression of contributions from higher-order sidelobes was mandatory for unambiguous axial superresolution. We show that novel lenses of 74 degrees semiaperture angle enable biological 4Pi microscopy with regular one-photon fluorescence excitation, thus increasing the signal and reducing system complexity and cost. An axial resolution of 95 nm, corresponding to a more than fourfold improvement over confocal microscopy, is verified in the imaging of microtubules in mammalian cells.

Characterization of Cep135, a novel coiled-coil centrosomal protein involved in microtubule organization in mammalian cells.

By using monoclonal antibodies raised against isolated clam centrosomes, we have identified a novel 135-kD centrosomal protein (Cep135), present in a wide range of organisms. Cep135 is located at the centrosome throughout the cell cycle, and localization is independent of the microtubule network. It distributes throughout the centrosomal area in association with the electron-dense material surrounding centrioles. Sequence analysis of cDNA isolated from CHO cells predicted a protein of 1,145–amino acid residues with extensive α-helical domains. Expression of a series of deletion constructs revealed the presence of three independent centrosome-targeting domains. Overexpression of Cep135 resulted in the accumulation of unique whorl-like particles in both the centrosome and the cytoplasm. Although their size, shape, and number varied according to the level of protein expression, these whorls were composed of parallel dense lines arranged in a 6-nm space. Altered levels of Cep135 by protein overexpression and/or suppression of endogenous Cep135 by RNA interference caused disorganization of interphase and mitotic spindle microtubules. Thus, Cep135 may play an important role in the centrosomal function of organizing microtubules in mammalian cells.

Isolation and characterization of the Golgi complex of the protozoan Trypanosoma cruzi

In this study the Golgi complex of the epimastigote forms of Trypanosoma cruzi were isolated and characterized. Using well-controlled sonication to rupture the cells and centrifugation on a discontinuous sucrose density gradient, a highly enriched Golgi fraction was obtained. The Golgi fraction contained most of the beta-galactosyltransferase (beta-Gal transferase) and UDP-N-acetyl-glucosamine: polypeptide-alpha-N-acetyl-glucosaminyltransferase (O-alpha-GlcNAc transferase) activities with minimal contamination of other organelles, as observed by enzymatic assays and electron microscopy analysis. To characterize the Golgi from T. cruzi cells further, it was incubated with a monoclonal antibody against a 58 kDa protein involved in the association of the Golgi complex with microtubules in mammalian cells. Immunofluorescence microscopy showed that the 58 kDa protein is localized in the T. cruzi Golgi region, a result confirmed by high resolution scanning electron microscopy immunocytochemistry. Thus, our results show, for the first time, that the beta-Gal transferase, the O-alpha-GlcNAc transferase and the 58 kDa protein are present in the Golgi complex of T. cruzi and are novel biochemical markers which can be used in the characterization of this organelle in T. cruzi.

Translation Initiation Factor eIF3 Probably Binds with Microtubules in Mammalian Cells

Association of the translation apparatus with the cytoskeleton is essential for its transportation within the cell and probably also for translation regulation. Very little is known about the involvement of particular proteins of this association. A polypeptide homologous with the heavy chain of translation initiation factor eIF3 p170 was found earlier in a microtubule preparation from adrenal cells. Antibody A167 directed against the recombinant fragment of p170 has been generated to study eIF3 interaction with microtubules in mammalian cells. This antibody was shown to recognize a single 170-kDa polypeptide in eIF3 preparations as well as in homogenates of various cell types. A167 allowed detection of the 170-kDa polypeptide in microtubule preparation from bovine brain and confirmation of its presence in microtubule preparations from adrenal cells. As shown by immunofluorescence microscopy using A167, the 170-kDa polypeptide is mainly located in the endoplasm within numerous small and some large granules. Cell treatment with cycloheximide resulted in growth and clustering of the large granules, and partial antigen redistribution along intracellular microtubules. These new experimental data indicate that mammalian translation factor eIF3 may bind with microtubules.

Cytoplasmic microtubules in mammalian cells

Cytoplasmic microtubules can be shown to be a constant finding in tissues of the rat which have been fixed in aldehydes followed by osmium tetroxide. The aldehydes employed were acrolein and glutaraldehyde, which were combined to give both rapid penetration and stabilization of the ultrastructural constituents of the cell. By this method microtubules are seen to be common to all areas of the cytoplasm. Connections to other membranes are illustrated. Possible functional roles for the cytoplasmic microtubules are discussed.