Structural and functional differences between porcine brain and budding yeast microtubules

The cytoskeleton of eukaryotic cells relies on microtubules to perform many essential functions. We have previously shown that, in spite of the overall conservation in sequence and structure of tubulin subunits across species, there are differences between mammalian and budding yeast microtubules with likely functional consequences for the cell. Here we expand our structural and function comparison of yeast and porcine microtubules to show different distribution of protofilament number in microtubules assembled in vitro from these two species. The different geometry at lateral contacts between protofilaments is likely due to a more polar interface in yeast. We also find that yeast tubulin forms longer and less curved oligomers in solution, suggesting stronger tubulin:tubulin interactions along the protofilament. Finally, we observed species-specific plus-end tracking activity for EB proteins: yeast Bim1 tracked yeast but not mammalian MTs, and human EB1 tracked mammalian but not yeast MTs. These findings further demonstrate that subtle sequence differences in tubulin sequence can have significant structural and functional consequences in microtubule structure and behavior.     

Microfilaments and microtubules: the news from yeast

New evidence that cortical actin patches and the endocytic machinery share components supports the idea that actin patches are in fact transient membrane coats at the initial stage of endocytosis. Recent studies of actin cables have identified formins as the core of a novel actin-filament-assembling machine. Meanwhile, microtubule-binding proteins have been found in the kinetochore, and factors affecting microtubule dynamic instability have been identified.     

Spc110p: assembly properties and role in the connection of nuclear microtubules to the yeast spindle pole body.

Spc110p is an essential component of the budding yeast spindle pole body (SPB). It binds calmodulin and contains a long central coiled-coil rod which acts as a spacer element between the central plaque of the SPB and the ends of the nuclear or spindle microtubules. This suggests that the essential function of Spc110p is to connect the nuclear microtubules to the SPB. To confirm this, we examined the phenotype of ts alleles of SPC110, one of which contains a mutation in the calmodulin binding site and was suppressed by overexpression of calmodulin. The alleles fail to form a functional mitotic spindle because spindle microtubules are not properly connected to the SPB. We also examined the phenotype of the toxic overexpression of either the wild-type or a truncated version of Spc110p containing a deletion of most of the coiled-coil domain. Both of these proteins form large ordered spheroidal polymers in the nucleus. The polymerization of the truncated Spc110p appears to be initiated inside the SPB from the position where Spc110p is normally located, and as the polymer grows in size it severs the connection between the nuclear microtubules and the SPB. The polymers were purified and are composed of Spc110p and calmodulin. A model for the structure of the polymer is proposed.     

Characterization of the peroxide sensitivity of COX-deficient yeast strains reveals unexpected relationships between COX assembly proteins

A number of distinct cuproproteins of the mitochondrial inner membrane are required for the assembly of cytochrome oxidase (COX), thought to function in a “bucket brigade” fashion to provide copper to the Cu_A and Cu_B sites. In yeast, the loss of two these proteins, Sco1p and Cox11p, leads to respiratory deficiency and a specific inability to survive exposure to hydrogen peroxide (H₂O₂). Using a quantitative assay, we have identified subtle differences in the peroxide-sensitive phenotypes between sco1 and cox11 mutant strains. Interestingly, the peroxide sensitivity of the sco1 null strain can be suppressed by overexpressing either SCO2 or COX11, although overexpression of neither SCO1 nor SCO2 can rescue the cox11 null strain. We also find that overexpression of either CTT1, encoding the cytosolic catalase T, or CTA1, encoding the mitochondrial matrix catalase, suppresses the peroxide sensitivity in both the sco1 and the cox11 null mutants. Direct measurement of peroxide metabolism shows that sco1 and cox11 null strains fail to degrade a significant amount of exogenously provided H₂O₂. Taken together, our data demonstrate that although Cox11p and Sco1p play distinct roles in COX assembly, they seem to play overlapping or related roles in peroxide metabolism that require further investigation.     

Essential role of tubulin-folding cofactor D in microtubule assembly and its association with microtubules in fission yeast.

The main structural components of microtubules are alpha- and beta-tubulins. A group of proteins called cofactors are crucial in the formation of assembly-competent tubulin molecules in vitro. Whilst an in vitro role is emerging for these cofactors, their biological functions in vivo remain to be established. In order to understand the fundamental mechanisms that determine cell polarity, we have screened for fission yeast mutants with altered polarity. Here we show that alp1+ encodes a homologue of cofactor D and executes a function essential for cell viability. A temperature-sensitive alp1 mutant shows a variety of defects including abnormal mitoses, loss of microtubule structures, displacement of the nucleus, altered growth polarity and asymmetrical cell division. Overexpression of Alp1 is lethal in wild-type cells, resulting in altered cell shape, but is rescued by co-overexpression of beta-tubulin. Alp1 co-localizes with microtubules, both interphase arrays and mitotic spindles. Furthermore, Alp1 binds to and co-sediments with taxol (paclitaxel)-stabilized porcine microtubules. Our results suggest that, in addition to a function in the folding of beta-tubulin, cofactor D may play a vital role in microtubule-dependent processes as a microtubule-associated protein.     

The spindle: a dynamic assembly of microtubules and motors

In all eukaryotes, a microtubule-based structure known as the spindle is responsible for accurate chromosome segregation during cell division. Spindle assembly and function require localized regulation of microtubule dynamics and the activity of a variety of microtubule-based motor proteins. Recent work has begun to uncover the molecular mechanisms that underpin this process. Here we describe the structural and dynamic properties of the spindle, and introduce the current concepts regarding how a bipolar spindle is assembled and how it functions to segregate chromosomes.     

cAMP inhibits the in vitro assembly of microtubules.

A new method for assaying microtubule assembly is described. The method utilizes the colchicine binding property of tubulin. This technique was used to study the effect of cyclic AMP on tubulin assembly using 100,000g supernatant and cycle-purified tubulin prepared from porcine brain. Cyclic AMP, in the presence of NaF, inhibited tubulin assembly from 100,000g supernatant but had no effect on cycle-purified tubulin.     

End-binding protein 1 stimulates paclitaxel sensitivity in breast cancer by promoting its actions toward microtubule assembly and stability

Paclitaxel is a microtubule-targeting agent widely used for the treatment of many solid tumors. However, patients show variable sensitivity to this drug, and effective diagnostic tests predicting drug sensitivity remain to be investigated. Herein, we show that the expression of end-binding protein 1 (EB1), a regulator of microtubule dynamics involved in multiple cellular activities, in breast tumor tissues correlates with the pathological response of tumors to paclitaxel-based chemotherapy. In vitro cell proliferation assays reveal that EB1 stimulates paclitaxel sensitivity in breast cancer cell lines. Our data further demonstrate that EB1 increases the activity of paclitaxel to cause mitotic arrest and apoptosis in cancer cells. In addition, microtubule binding affinity analysis and polymerization/depolymerization assays show that EB1 enhances paclitaxel binding to microtubules and stimulates the ability of paclitaxel to promote microtubule assembly and stabilization. These findings thus reveal EB1 as a critical regulator of paclitaxel sensitivity and have important implications in breast cancer chemotherapy.     

Controlled clockwise-counterclockwise motion of the ring-shaped microtubules assembly

The microtubule (MT)-kinesin system has been proposed as the building block of biomolecular motor based artificial biomachines. Considerable efforts have been devoted to integrate this system that produced a variety of ordered structures including the ring-shaped MT assembly which is being considered as a promising candidate for the further development of the biomachines. However, lack of proper knowledge that might help tune the direction of motion of ring-shaped microtubule assembly from counterclockwise to clockwise direction, and vice versa, significantly restricted their potential applications. We report our success in controlling the direction of rotational motion of ring-shaped MT assembly by altering the preparation conditions of microtubules. The change in the direction of rotation of MT rings could be interpreted in terms of the accompanied structural rearrangement of the MT lattice. For achieving handedness-regulated efficient biomachines having tunable asymmetric property, our study will be significantly directive.     

Production of human type I collagen in yeast reveals unexpected new insights into the molecular assembly of collagen trimers

Substantial evidence supports the role of the procollagen C-propeptide in the initial association of procollagen polypeptides and for triple helix formation. To evaluate the role of the propeptide domains on triple helix formation, human recombinant type I procollagen, pN-collagen (procollagen without the C-propeptides), pC-collagen (procollagen without the N-propeptides), and collagen (minus both propeptide domains) heterotrimers were expressed in Saccharomyces cerevisiae. Deletion of the N- or C-propeptide, or both propeptide domains, from both proalpha-chains resulted in correctly aligned triple helical type I collagen. Protease digestion assays demonstrated folding of the triple helix in the absence of the N- and C-propeptides from both proalpha-chains. This result suggests that sequences required for folding of the triple helix are located in the helical/telopeptide domains of the collagen molecule. Using a strain that does not contain prolyl hydroxylase, the same folding mechanism was shown to be operative in the absence of prolyl hydroxylase. Normal collagen fibrils were generated showing the characteristic banding pattern using this recombinant collagen. This system offers new opportunities for the study of collagen expression and maturation.     

Mitochondria, molecular chaperone proteins and the in vivo assembly of microtubules

Two proteins, P1 and P2, which are specifically altered in mammalian cell mutants resistant to antimitotic drugs, have been identified as the homologs of two members of the class of proteins known as molecular chaperones. P1 is localized in mitochondria and P2-related proteins are involved in the translocation of proteins to mitochondria. To account for these and a number of other observations, a new model for in vivo microtubule assembly is proposed.     

Effect of guanine nucleotides on the assembly of brain microtubles: ability of 5'-guanylyl imidodiphosphate to replace GTB in promoting the polymerization of microtubules in vitro.

Polymerization of microtubule requires the presence of GTP, and the tubulin-bound GTP is hydrolyzed during microtubule formation. However, it was found that an unhydrolyzable analog of GTP, 5′-guanylyl imidodiphosphate (Gpp(NH)p), was able to replace GTP. The hydrolysis of the terminal phosphate group of GTP, therefore, does not seem to be a prerequisite to $\text{in vitro}$ assembly of microtubules. The microtubules formed in the presence of Gpp(NH)p were indistinguishable from those formed in the presence of GTP under electron microscopy, but a remarkable decrease was noted in their sensitivity to depolymerization by calcium ions.     

Effects of ultraviolet light on the in vitro assembly of microtubules

Exposure of microtubular protein to ultraviolet light inhibits its assembly into morphologically normal microtubules. This effect appeared to result primarily from damage to the tubulin dimers. The damage consisted of a conformational change, a loss of two free sulfhydryl groups, a production of higher molecular weight cross-linked species, and the formation of aggregated amorphous material upon polymerization.     

The stabilization of microtubules in isolated spindles by tubulin-colchicine complex

We have analyzed the effect of colchicine and tubulin dimer-colchicine complex (T-C) on microtubule assembly in mitotic spindles. Cold- and calcium-labile mitotic spindles were isolated from embryos of the sea urchin Lytechinus variegatus employing EGTA/glycerol stabilization buffers. Polarization microscopy and measurements of spindle birefringent retardation (BR) were used to record the kinetics of microtubule assembly-disassembly in single spindles. When isolated spindles were perfused out of glycerol stabilizing buffer into a standard in vitro microtubule reassembly buffer (0.1 M Pipes, pH 6.8, 1 mM EGTA, 0.5 mM MgCl2, and 0.5 mM GTP) lacking glycerol, spindle BR decreased with a half-time of 120 s. Colchicine at 1 mM in this buffer had no effect on the rate of spindle microtubule disassembly. Inclusion of 20 microM tubulin or microtubule protein, purified from porcine brain, in this buffer resulted in an augmentation of spindle BR. Interestingly, in the presence of 20 microM T-C, spindle BR did not increase, but was reversibly stabilized; subsequent perfusion with reassembly buffer without T-C resulted in depolymerization. This behavior is striking in contrast to the rapid depolymerization of spindle microtubules induced by colchicine and T-C in vivo. These results support the current view that colchicine does not directly promote microtubule depolymerization. Rather, it is T-C complex that alters microtubule assembly, by reversibly binding to microtubules and inhibiting elongation. In vivo, colchicine can induce depolymerization of nonkinetochore spindle microtubules within 20 s. In vitro, colchicine blocks further microtubule assembly, but does not induce rapid disassembly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of spirogermanium on the in vitro assembly and disassembly of brain microtubules

The inhibition of microtubule proteins (MTP) assembly by Spirogermanium (SP, 1.25-100 microM) has been studied. Assembly at 37 degrees C was monitored by turbidity measurements and electron microscopy. For SP in 1:1 protein-drug ratio the inhibition of assembly was 50%. Addition of 12.5 microM SP to microtubules induced spontaneous disassembly. SP had less effect on the assembly of pure tubulin (tubulin 6S). Complete inhibition of assembly induced by glycerol and Mg2+ was found with 250 microM and the ratio of SP to tubulin to obtain 50% inhibition was higher than with MTP.     

Cold depolymerization of microtubules to double rings: geometric stabilization of assemblies

The kinetic pathway of microtubule depolymerization at 0 degrees C has been examined. Microtubules made of MAP-containing and MAP-free tubulins were depolymerized at 0 degree C in the presence of [3H]GDP or [3H]GTP or of trace amounts of 125I dimeric tubulin. The products of depolymerization were separated on a column, their structures were identified by electron microscopy, and the time course of incorporation of 3H or 125I labels in the different components of the system was determined. Two predominant assembly states of tubulin found in the nonmicrotubule state were alpha-beta dimers and double rings. Kinetic data indicate that ring formation from disassembling microtubules does not occur by direct coiling of protofilaments as previously thought, but disassembling GDP subunits are in very rapid equilibrium with curved oligomers that are kinetic intermediates in the isodesmic assembly of GDP-tubulin. The formation of oligomers and rings from dimers, at concentrations as low as 10 microM, is much faster than nucleotide exchange on alpha-beta-tubulin. Disassembly of double rings, in contrast, is slower than nucleotide exchange on alpha-beta-tubulin, by 1 order of magnitude in the absence of MAPs and 2 orders of magnitude in the presence of MAPs. These results support the model proposed previously to explain spontaneous oscillations in microtubule assembly. They are consistent with the existence of an equilibrium between two conformations of tubulin, "straight", i.e., microtubule forming, and "curved", i.e., ring forming, under the allosteric control of bound nucleotide. The straight conformation requires the presence of two ionizable hydroxyls on the gamma-phosphate in GTP or GDP-Pi.     

Suppression mechanisms of COX assembly defects in yeast and human: insights into the COX assembly process

Eukaryotic cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial respiratory chain. COX is a multimeric enzyme formed by subunits of dual genetic origin whose assembly is intricate and highly regulated. In addition to the structural subunits, a large number of accessory factors are required to build the holoenzyme. The function of these factors is required in all stages of the assembly process. They are relevant to human health because devastating human disorders have been associated with mutations in nuclear genes encoding conserved COX assembly factors. The study of yeast strains and human cell lines from patients carrying mutations in structural subunits and COX assembly factors has been invaluable to attain the current state of knowledge, even if still fragmentary, of the COX assembly process. After the identification of the genes involved, the isolation and characterization of genetic and metabolic suppressors of COX assembly defects, reviewed here, have become a profitable strategy to gain insight into their functions and the pathways in which they operate. Additionally, they have the potential to provide useful information for devising therapeutic approaches to combat human disorders associated with COX deficiency.