Molecular Motors, Filaments and Cell Dynamics/ Moteurs moléculaires, filaments et dynamique cellulaire

Symposium organized by Éric Kаrsenti and Michael Sheetz, from 7- 13 mars 2000.

 

Participants

Viki ALLAN (University of Manchester, Royaume-Uni), Chloé BULINSKI (Columbia University, New York), Larry GOLDSTEIN (Howard Hughes Medical Institute (H.H.M.I.), La Jolla, USA), Bruno GIUD (U.M.R. CNRS 144, Institut Curie, Paris), Steve GROSS (University of California, Irvine, USA), Eric KARSENTI (European Molecular Вiology Laboratory (E.M.B.L.), Heidelberg, Allemagne), Kazuhiko KINOSITA (Tokyo University, Kawasaki), Daniel LOUVARD (Institut Curie, Paris), Dietmar J. MANSTEIN (Max-Planck Institute for Medical Research, Heidelberg Allemagne), François NEDELEC (European Molecular Biology Laboratory (E.M.B.L.), Heidelberg, Allemagne), Jacques PROST (Institut Curie, Paris), Michael P. SHEETZ (Columbia University, New York), Jonathan M. SCHOLEY (University of California, Davis, USA), Robert H. SINGER (Albert Einstein College of Medicine, Bronx, USA), James A. SPUDICH (Stanford University School of Medicine, USA), Richard VALLEE (University of Massachusetts, Worcester, USA), Isabelle VERNOS (European Molecular Biology Laboratory (E.M.B.L.), Heidelberg, Allemagne), Michael WAY (European Molecular Biology Laboratory (E.M.B.L.), Heidelberg, Allemagne), Marino ZERIAL European Molecular Biology Laboratory (E.M.B.L.), Heidelberg, Allemagne).

 

SUMMARY

The goal of the meeting on Molecular Motors, Filaments and Cell Dynamics was to bring together experts from this field work­ing on aspects ranging from the molecular mechanism of force pro­duction by rotors moving along the fibers present in cells (micro-tubules and microfilaments) to the effects of such systems on cellular activities, shapes and functions. The idea was to give a long time to each speaker in order to have informal talks, open to free questions, to generate lively sessions in the format of a brain­storming, rather than a classical meeting. This was a great success.

Movements in cells have been observed since the very beginning of the field of Cell Biology and the advent of light microscopy. How those movements are produced has remained an enigma, until the discovery of the existence of cytoplasmic molecular rotors that could rive along filaments made of actif or micro-tubules. Actif filaments have been known for a long time, as thеу form the thin filaments (6nm) of muscle cells. Together with the motor myosin, thеу are responsible for the generation of force bу muscles. A lot of work has been dedicated towards the under­standing of how myosin could transform the chemical energy pro­duced by the hydrolysis of ATP into mechanical energy.

Although mechanical details have been obtained, the exact mechanism remains a malter of debate. In other cells, actif filaments play also an important role in cytoplasmic organization, motility dynamics and functions, in conjunction with a variety of myosin variants. Microtubules are hollow tubes, 25 nm in diameter and built of 13 protofilaments. They participate in the formation of many cellular structures, ranging from beating cilia and flagella to the mitotic spindle during mitosis. In interphase cells they organize the cyto­plasm and, in particular, direct the movement of vesicles and organ­ization of the membrane trafic maсhinery. The rotors associated to microtubules belong to two large categories: those related to dynein on one hand, and to kinesin on the other. Microtubules are oriented polymers with a “plus” and a “minus” end.

The plus end is highly dynamic, undergoing frequent transitions between phases of growth and shrinking. The minus en is that which forms first when a microtubule grows from tubulin subunits. This end is rela­tively inert once it has formed. Microtubule rotors can read this polarity on the surface lattice of microtubules. Dyneins are minus end rotors and most kinesins, with a few exceptions, are plus end directed rotors. There is a huge diversity of kinesins and much less of dynein. The variety in the kinesin subfamilies comes mostly from the structure of the tails, that are used as specific devices to provide each motor with a specific “function” in the tell by target­ing the motor domain to a specific organelle. Some are targeted to vesicles, others are targeted to chromosomes, other still are used to organize microtubules themselves in various patterns and machines. The biggest mystery associated with living systems con­cerns the emergence of complex structures from a complex mixture of components. Filament/motor systems are good problems to study in this context. Among other things, like the mitotic spindle that is used to segregate chromosomes, rotors and their associated filaments can also make sensory organs, like those present in the cells from the inner car. Also, at the opposite end, there are rotors that are used to produce energy in the cell – just like an alternator, moved by a diesel engine, can make electricity. In the cell, there are rotors that produce the fuel of life, ATP, from electrical gradients (proton gradients). The coupling between these processes is just amazing. The invited speakers have been discussing recent results and ideas about all these aspects of the rotors systems.

 

REPORT

Motor structure and mechanism of force production

Three speakers addressed these issues, Manstein, Spudich and Vallee. Manstein addressed the question of how the structure of ATP binding domains affect the motility properties of myosin on actif filaments. This was based on the Tact that nucleotide motifs in G proteins and rotors are highly conserved 7 β sheets and 6 helices. Results show that Myosín switch 2 helix rives converter domain and produces displacement. Relay loop mutant give a dominant negative phenotype myosin. Tryptophan fluorescence can be used to measure changes in ATP hydrolysis. RLP did not show hydrolysis. Removal of N-term SH3 domain does not allow rescue of full cells (N-term 40 aa deletion produces dead myosin). One can substitute other sequences in N-terminal and restore the аctivity. R238Е and E459R double mutation is active, whereas individual mutations are nearly dead. The double mutant has much lower ATPase activity and rives at 0.4 vs.1.0 microns/sec.

Actin binding domain structure, and secondary actin binding domain, are both bound. Changing charges in the secondary bind­ing domain has little effect, but insertion of mutations in loop 2 increases stability. ATP stabilizes myosin from unfolding (it has a steeper denaturation curve). Spudich reported data concerning properties of myosin family rotors in vitro and how they work. There were two parts in the presentation. One dealt with myosin movement along actif in general and the other with mechanical and physical properties of a specific myosin – Myosin V

Myosin movement

Removal of all of the cysteines from the heavy chain followed bу the addition of on back in appropriate location; also having only one cysteine in the light chain results in а change from а 4 to 7 nm step, when going from prestroke (ADP and vanadate) to rigor (ADP aton). ATP hydrolysis is needed to go into prestroke state. The сусlе involves ADP-Pi to ADP with Pi release for the conformation change of myosin to occur. ATP then binds back and myosin goes again into the prestroke state. Myosin binds to actif for 2 ms for а 10 nm displacement, which gives а speed of move­ment of about 5 microns per second.

Dual beam optical trap experiments, involving the capture of beads bound to actin filaments to bring it near а large bead coated with myosin, were carried out. This allowed seeing myosin movement along the actin filament. It was found that there is а decrease in the Brownian motion of the beads when myosin binds to the filament. It was also found that the mean displacement of myosin along the filament is about 7 nm, with а mean binding life­time inversely proportional to ATP concentration. This will allow using the change in Brownian motion as а criterion to define attachment events.

Myosin V

Myosin V is involved in intra-cellular mеlanoсyte transport. Mutants in Myosin V produce a Neurological defect in mice (Griscelli’s syndrome in humans). Myosin V in vitro moves single actif filaments on glass. Up to 15 microns of displacement were seen. Myosin V was put on 0.35 micron beads and placed on actif filaments. This allowed seeing a 36 nm displacement with a stalling force of 2.5 pN. A feedback system was used to hold the force con­stant as the motor moves. An estimated displacement of about 38 nm was found, even at forces of 1.8 pN and this did nit change with ATP concentrations. Off-rate for ADP is measured to be 13 per sec. High load situations lead to half (18 nm steps) steps that can either go forward or backward with a longer time constant.

Vallee reported on the structure of the microtubule motor Cytoplasmic Dynein; its binding properties to microtubules and correlations with diseases.

Structure

Cytoplasmic Dynein has 7 different subunits. There are only 2 cytoplasmic dyneins (CD) in the genome and Only 1 in a given cell. The lotir domain is 350 kDa while the whole protein is 500 kDa. The stalk (12-14 nm) is the micritubule binding domain (4 P loops are required forming 4 binding sites). Stalks have high alpha helical content. The motor domain (350 kDa) contains the ATPase activity. The 2nd CD is homologous but different in major ways from normal CD. Interaction sites between heavy chains and light intermediate or intermediate chains are found in base domain. A Stoechiometry of 2 heavy chains, 2 light chains, and 2 intermedi­ate chains plus some light chains was found per active molecule.

CD Functional Binding

There are many functions that involve CD and this involves manу components that probably produce different cargo interac­tions. The Dynactin complex (p150, p62, p50, Arp1, capping pro­tein, p27, p24 and Arp11) is involved in this and it is about the same size as CD. One protein of the dynаctin complex, called p50 or Dynamitin causes disruption of the dуnactin complex, disrup­tion of the Golgi and arrest cells in prometaphase. Dynamitin binds to kinetochores and Pericentrin was no longer concentrated at cen­trosomes in the presence of excess dynamitin in cells. Light chain 1 (not 2) binds to pericentrin in vitro and Pericentrin overexpression in cells gives a dynamitin phenotype. Virus infection (vaccinia) causes similar phenotype.

CD Disease Models

Lis-1 (Lisencephally) is related to Nudf (CD components in aspergillus). The genetíc disease Lisencephally shows major reori­entation of the brain. Layers are lost in the brain. In this case, nuclear migration is inhibited in the brain and not in muscles. PAFAH subunit (platelet activating factor acetylhydrolase) is related to LIS-1. And CD-dynactin complexes can be co-immuno­precipitated with anti-LIS-1 antibodies. Overexpression of LIS-1 causes increase in mitotic index and causes hairy microtubules and large microtubule arrays. LIS-1 does not co-purify with native CD from the brain. The plus end microtubule binding factor EB 1 stays, but dynactin is knocked off MT ends in LIS-1. Anti-LIS-1 anti­bodies knock chromosomes off the metaphase plate.

Motors and Cellular Transport

Eight speakers addressed this topic: GoLdstein, Goud, Gross, Singer, Allan, Way, Louvard and Sheetz. Goldstein reported on the role of microtubule orientation, Kinesins and cytoplasmic dynein in vesicle and microtubule transport in neuronal cells, as well as Golgi apparatus organization in epithelial cells. Microtubule orien­tations are important for directing motion. In neurons, micro-tubules have their plus ends toward the peripheryin axons, whereas mixed populations of microtubules exist in dendrites. In both cases, microtubule arrays are produced by the transport of microtubules either by K1р II or CD. In ciliary transport too, kinesin is impor­tant. For example, a KinII homologue (FLA 10, KIF3A, B, C and КАР) is required for transport of raft complexes. In mice lacking KIF ЗА, embryos lack cilia and display a situs inversus phenotype. Mutants in KIF3A in photoreceptor cells accumulate veasicles at the connecting cilium of these cells, while other cells are normal. Therefore, KIF3A is clearly important for transport of important membrane components in cilia and their morphogenesis.

Goud reported that a Rab protein interacts with a kinesin and that this is involved inn ER Golgi transport. A dominant negative mutant of Rab6, stick in а GTP form, induces the Golgi apparatus to retract back into the reticulum and salutatory movements of vesicles along microtubules are seen from the Golgi. It is not clear ехасtlу how Rab6 functions in this process, or what the function of Rab kinesin is, but this motors seems to interact with Rab6 through a coiled coil domain. Gross addressed the question of bi-directional move­ment of vesicles along microtubules.

How is net transport regulated?

Saltatory movements in cells along microtubules show bidirec­tional motion. But it is a question of whether the motors are alter­nating by motor activation, or interaction with various vesicles.

Clearing of lipid droplets in insect embryos is found in many insect embryos at early stage 14 (cloud at late stage 14) and some of the mutants in droplet clearing do not have other problems. In Drosophila embryos, for example, the + ends of the MTs are at the center, whereas — ends are at the membrane. The minus end move­ment of droplets are CD dependent and the plus end movements are driven by kinesin-like proteins. Velocities are 0.5-1 micron/sec. Lipid droplets don’t have a bilayer but some proteins do bind. Average run length is 800 nm towards the + end and 600 nm towards the — end in clearing phase. They are 400 nm towards the + end and 600 nm towards the — end in the clouding phase. Therefore, the 2 phases of movement are generated by short slow runs and faster long rues. The length of long, faster runs changes when the net transport direction changes.

How manу active motors are on droplets?

The measure of stall force was used to estimate the number of rotors per vesicle. However, there was a range and it was therefore necessary to use the percentage of droplets that escape at a given force. Mean stall force increases in clearing from 3 to 5 pN, whereas it is 4 pN in clouding. Particles resume movement after stalls. One gets the same average stall forces in both directions. This suggests that there are about 4 rotors per droplet and that reversibility is not related to processivity (since there are multiple rotors and pauses are not observed between reversais). Glued mutant embryos show decreased mn length in — end direction, but no change in velocity or stall force. Glued mutation causes unreg­ulated binding of CD to MTs. In this case, one seer a dramatic decrease in + end travel length and a decrease in stall force in the plus direction. CD mutation also has a drop in force.

Klar mutant has a much lower stall force (the mutant is charac­terized by lack of clouding). There is also a dramatic decrease in run length in the plus direction. Klar is a 250 kDa protein with few homologies and does not copurify with droplets or rotors.

Sheetz addressed the question of Myosin V binding to brain vesicles. Myosin II and V are important for the generation and seg­regation of subcellular compartments. Vesicular myosin II and V are found associated with the protein scaffolding of a common sub-set of vesicles by density sedimentation, electron microscopy, and immu-nofluorescence. Solubilization of either myosin II or V was caused by polyphosphates with the following efficacy at 10 ml for myosin II; ATP-Mg 2+ = ATP = AMP-PNP (5*-adenylyl imi­dodiphosphate) > pyrophosphate 5 tripolyphosphate > > tetrapolyphosphate 5 ADP > cAMP = Mg 2+ ; and for myosin V pyrophosphate = tri-polyphosphate > ATP- Mg 2+ = ATP = AMP-PNP > > ADP = tetrapolyphosphate > cAMP = Mg 2+ Consequently, solubilization is not an effect of phosphorylation, hydrolyeis, or disassocia­tion of myosin from actin filaments. Scatchard analysis of myosin V binding to stripped dense vesicles showed saturable binding, with a Km of 10 nM. Analysis of native vesicles indicates that these sites are fully occupied. Together, these data show there are over 100 myosin Vs/vesicle (100-nm radius). It is proposed that polyphosphate anions bind to myosin II and V and induce a con­formational change that disrupts binding to a receptor. Another aspect of the Sheetz report dealt with a completely different story: that of Plus retraction in bacteria and how this could power bac­terial twitching and motility. Twitching and social gliding motility allow many Gram negative bacteria to crawl along surfaces, and are implicated in biofilm formation, signaling to host cells, and viru­lence. Type IV pili (Tfp) are required for twitching and social glid­ing, but the mechanism by which these filamentous appendages promote motility has been enigmatic. They have used the laser tweеzers67 to demonstrate that Tfp can forсefully retract. Tfp­producing Neisseria gonorrhoeae cells activеly crawl on a glass sur­face and form adherent microcolonies. When laser tweezers are used to place aid hold cells near a microcolony, the cells are pulled from the laser trap toward the microcolony. In quantitative experi­ments, latex beads bind to the Tfp of immobilized cells and are pulled from the laser trap toward immobilized cells. Individual diplococci exert tensile forces of >80 pN on 1 µm beads. Like twitching motility, Tfp retraction is intermittent, occurs at 1 µm s-1, requires protein synthesis and depends on PilT function. Their experiments establish Tfp as a novel class of nanomotors capable of generating substantial retractile force.

Allan addressed the question of regulation of the interaction of rotors with membranes and the importance of this in the regula­tion of the direction of vesicle transport. In the ER to Golgi mem­brane trafic, vesicles budding from the ER rive to the Golgi in a CD dependent way. The plus end directed movement from the Golgi back to the ER is inhibitied by the Hl anti-kinesin antibody. There might be some regulation of the relative binding, or relative aсtivity of these two rotors between the two pathways. During mitosis, ER movements are inhibited by more than ten times; while the CD dependant movement of beads is not altered. The amount of membrane-bound CD and dynactin is decreased seven fold in moitosis and there is а 12 fold increase in phosphorylation of the light intermediate chain of dynein. Intermediate chain and dynactin phosphorylation are seen at earlier changes in cell сус1е. Which kinase is responsible for activity? There is an increased phosphorylation induced by the сdс2 kinase. Vale correlated this with the fact that the 5197А mutation blocks phosphorylation. The сdс2 kinase causes а release of CD from membranes in vitro. Dynactin comes off as well.

Singer addressed the problem of the role of messenger RNA Localization in Cell Polarity and Embryo Development.

β-actin mRNA is localized to the leading edge of the cell near the lamellipodia. А ‘zip code’ in the 3′ UTR contains sequences that determine the localization of mRNA. Disruption of the zip code by anti-sense, results in loss of spatial localization of transla­tion in four hours and cеlls assume an epithelial as opposed to fibroblastic morphology. Cell migration is inhibited but not severely altered. Persistence of migration is inhibited most dramat­ically by the loss of localization. Actif polymerization occurs but without polarity at the leading edge. Serum deprivation results in loss of actif mRNA asymmetry and addition of serum results in estabishing asymmetry in 5-10 minutes. Constitutively active Rho keeps mRNA localized in the absence of serum and inactive Rho will prevent localization of message in response to serum. Latrunculin blocked redistribution and there was less loss of asym­metry than with nocodazole. Н7 and BDM, myosin inhibitors, both inhibit the localization of message. Myosin IIB knockout fibroblasts (from Adelstein at NIH) dо not show localization of mRNA in response to serum and dо not polarize well. Other mes­sages related to actif dynamics appear to be localized as well. ER does not appear to be involved in localization and probaby not in rapidly moving cells such as Dictyostelium, although little is known about localization in these systems.

Highly asymmetric cells such as fleurons have MT-dependent transport of mRNA. ZBP1 binds to the zip code mRNA sequence, and has an NLS and nuclear export signal. Antibody staining shows that it is at the leading edge of fibroblasts. High resolution microscopy shows that it can align along actin stress fibers and MTs. Zipcode antisense delocalizes both mRNA and ZBP1, show­ing it is an RNA-protein complex, which is necessary. Leptomycin B (inhibitor of crim1 dependent export from nucleus) causes buildup of ZBP1 in the nucleus. ZBP1 goes onto mRNA at the time of transcription. Veg1 localization sequence from Xenopus binds a protein which is the ZBP1 ortholog. ZBP1 can be overex­pressed in many cancers.

Localization in yeast of Ash1 mRNA is seen in the bud. Five mutants disrupt the RNA localization including type V mуosin (myosin IV). Stem-loop-bulge is required for localization (struc­ture and not sequence dependent binding) . A single RNA-depend­ent particle is formed in yeast, and that particle moves to the bud. The particle rives in a saltatory fashion (move-stop-diffuse-move) from the mother cell to the bud.

Louvard examined how actin meshworks could be generated in cells, and found two different mechanisms.

ActA bacterial protein is necessary for Listerial actif filament tal generation and movement. The protein is composed of two subdomains : a 234 aa N-terminal domain that contains actin nucleation aсtivity, and a 380 aa proline-rich C-terminal domain. The activity of ActA requires host cell proteins as shown by exper­iments in which expressions of ActA in cells (without Listeria) drives actin polymerization. In one type of experiment, ActA can be engineered to include a CAAX box that will cause it to insert at the inner plasma membrane, which is enriched in actin regulatory proteins. ActA-CAAX induces the production of many long, actin­rich projections from the cell surface. When human cells are stained with anti-ActA antibodies, focal adhesions are detected, suggesting that a homolog is present at these actin rich sites. The cellular anti­gen was identified, in collaboration with Mary Beckerle, as ZyXin an 84 kDa protein composed of a proline rich N-terminal domain, a nuclear export sequence and 3 LII domains. The N-terminal domain-shares 23% sequence identity with the proline rich C-ter­minal domain of ActA, whereas the other Zyxin domains are not present in the ActA sequence. LPP (Lipoma preferred partner) is a protein that is structurally similar to zyxin. Zyxin binding partners include Vav, VASP, alpha-actinin, and cysteine rich protein (CRP).

To understand how cells polymerize actin, one can use the infor­mation acquired from the Study of Listeria movement using puri­fied protein mixtures. ActA can bind VASP and the Arp2/3 com­plex to stimulate actin polymerization. In order to make the polymérisation productive in terms of movement, filaments must be capped at the barbed ends and ADF/cofilin drives depolymeri­sation and reactivation of ATP/G-actin. The cellular homologues to N-terminal domain of ActA (known as the nucleator) are members of the WASp family of proteins. One can use human WA subdo­main (99 aa present in the C-terminus, also known as VCA) from WASp as a core actin polymerization unit. WA coated latex beads will assemble actin in vitro when placed into a HeLa cell-free extract that contains actin labeled with rhodamine. These beads generate comets and move, despite the fact that they do not con­tact an F-actin binding site which is thought to be essential for movement. Another actin polymerization system has been identi­lied by testing the zyxin-like (proline rich) C-terminal region of ActA. When placed in extracts, Pro beads will polymerize actin, but, when viewed by electron microscopy, they display a filopodial­like actin bundle instead of the orthogonal networks produced by WA beads. Soluble WA does not affect polymerization on Pro beads, but will block polymerization on WA beads. Pro does not bind the Arp2/3 complex, but does bind VASP (which is present in focal adhesions) suggesting that these are two, independent actin polymerization systems, both of which are present in ActA.

Another method to test actin polymerizаtion activity is to ectopi­cally target candidate proteins to mitochondria so that thеу remain in a cellular context while amenable to experimental manipulation. Zyxin-mito proteins were prepared by engineering variants with the ActA membrane anchor, and transfecting cells with cDNAs encoding these variants. Cells were permeabilized with saponin and fluorochrome tagged G-actif was added to follow actin polymer­ization. Full-length zyxin, and N-term zyxin (that contains the ActA-like domain) assembled actin rich structures onto mitochon­dria, whereas the LIM domains of zyxin did not display this activ­ity. Alteration of 4 critical residues in the proline rich region of Zyxin-mito, which block Zyxin/VASP interaction, prevented G-actif incorporation at mitochondria. The addition of a peptide, LМEEMNАMLARRRKAT, which mimics a C-terminal domain of VASP, blocked actin polymerisation, while a peptide that dis­rupts VASP/profilin interaction, had no effect.

Way examined also the mechanism of actin meshwork assembly when viruses infect cells. Indeed, it is known that when Vaccinia Virus infects cells, it induces Actin Tails as listeria does. Suсh actin tails form only at the bottom of the cell. Vaccinia virus is sequenced, and codes for 260 proteins (about 90% are known in function). The core of the virus binds to MTs by viral MAPs and ends up at the Golgi during infection. IMV (intracellular mature virus) has no actin tail but it does move out. IEV (intracellular envelope virus) takes up a double membrane from the TGN. Infectious particles have 2 or 3 membranes around them. VASP is localized to the virus but nit zyxin. No profiling is present. Phosphorylated tyrosine residues (P-tyr) are found on VASP in vaccinia but not listeria or shigella actin tails. Micro-injection of P­tyr Ab inhibits actin tail formation. Vaccinia proteins, A34R aid A36R are needed for actin tail assemЫу. A36R rescues actin tail formation and tyrosine 112 of A36R is needed.

Type 1 phosphatase inhibits tail formation. Dominant negative Src is recruited to actif tans. Srс, Fyn and Yes minus cells still support tail forma­tion. Nck and N WASP are both recruited to viral particles. Nck­SH2 will not bind in vivo but will in vitro. This suggests that A36R binds Nck then N WASP and then Arp2/3. N WASP has PH domain, calmodulin, Cdс42, SH3 adaptors, actif, and Arp2/3. On1y WH1 domain binds to the virus (contains PH and IQ). Cdc42 but not Rac is recruited to the virus tail but is not needed for tail formation. WASP (Wiskott-Aldrich Syndrome Protein) is only expressed in hematopoietic cells and mutants are in WH1 domain. N WASP is universally expressed and mutant forms do not support tail formation of the virus. WIP is recruited to actin tans. GFP-WIP-WBD (WASP-binding domain) inhibits tail forma­tion. This now suggests that A36R then Nck binds WIP and N­WASP and then Сdс42 plus Arp2/3. Cell morphology is dramati­сally altered by the virus (spreads and rives faster) and one hour alter infection the centrosome is lost.

Motors and Mitosis

Four speakers addressed this issue: Karsenti, Scholey,  Vernos and Nedelec. Karsenti addressed the general question of Self organization in cell and organ shape. The general idea is that, with the dynamies of building blocks, it is possible to generate complex patterns and that, with added rotors, it is possible to obtain polar­ization aid transport. The problem of spindle morphogenesis was treated in some more details. Spindles are always bipolar whether centrosomes are present (Mitosis) or not (Meiosis). Xenopus egg mitotic extracts make no microtubules in the absence of centro­somes and short dynamic microtubules off centrosomes. XMAP215 and 1AP230 stabilize MTS whereas stathmin or XKCM1 destabilize them. X1AP230 prevents catastrophes but affects only slightly the growth rate, whereas MAР215 increases growth rate but has little effect on catastrophes in pure tubulin solutions. However, in egg extracts, XMAP215 opposes the catas­trophe inducing activity of XKCM1. In the absence of XMAP215, the spindles formed are very small. The local stabilization of microtubules near DNA beads has been seen, but there is no quantita­tion yet. When microtubules meet a chromatin bead, thеу are captured. This suggests there is a local regulation of MT dynamics. The hypothesis is that stathmin is more phosphorylated by chro­matin, leading to its inactivation and a more stable population of microtubules around chromosomes. Many kinases on chromatin.

Microtubules are nucleated around chromosomes (DNA) with their minus ends awаy. This is induced by the local generation of RanGTP at chromosomes, because of the presence of RCC1 on chromosomes. Factors involved in this process are being purified.

RanT24N (GDP or еmpty form) blocks the nucleation of microtubules by chromosome but not from centrosomes. There is no effect on interphase microtubules. Ву contrast, RаnQ69L-GTP induces MT assembly in mitotic extracts in the absence of chromo­somes. Once microtubules are nucleated and stabilized, theу are organized in a bipolar array by various rotors that cross-link and orient microtubules.

In summary, there are 2 Morphogenetic Principies in cells: Stereospеcificity and Self-organization that involves as one compo­nent, rotors. There seems to be a Morphogenetic field around chromosomes made of G-protein gradient, phosphorylation gradient and motor action on polarized MTs. Centrosomes are not essential even in mitotic cells (UV irradiation can destroy centrosomes but not spin­dle assemby) and a Drosophila line grows without centrosomes. It seems that under physiological conditions, chromatin is the major driving force to initiate spindle formation and to determine the final size and bipolar organization of the spindle, in conjunction with a global regulation of MT dynаmics during the interphase to metaphase transition.

Vernos addressed more specifiсally the role of two chromosomal motors (Xkid and XKlp1) in stabilizing the bipolar spindle and in positioning chromosomes on the metaphase plate. Xkid is closely related to human kid at the sequence level. Xenopus and human kid have two predicted helix-hairpin-helix DNA binding domains at their C-terminus, and are associated to chromosome arms during mitosis. The function of Xkid was exam­ined by looking at spindle аssembly in Xkid depleted Xenopus egg extracts. Microtubules bесamе organized into bipolar spindles but chromosomes did not align on the metaphase plate and appeared to stretch all along spindle microtubules. Anti-XCENP-E immuno­fluorescence on spindles assembled in Xkid depleted extract shows that kinetochores are stil paired. Although many of them are found around the spindle equator, some of them are at the proximity of spindle piles. Addition of anti-Xkid antibodies to extracts interferes with Xkid localisation to chromosomes and produces a similar phenotype. Tо test if Xkid function is still required after chromo­some alignment, antibodies were added to preformed spindies with chromosomes aligned on the metaphase plate. Chromatin rods, presumably chromosome arms, moved towards spindle poles, gen­erating progressively the same chromosome misalignment pheno­type found in depleted extracts. This indicates that Xkid is, at least in part, responsible for the so-called polar ejection forces that move chromosome arms towards microtubule plus ends during mitosis. This аctivity is essential to ensure a tight alignment of chromo­somes on the metaphase plate and to maintain this alignment dur­ing metaphase. In addition, this provides a ‘robust’ mechanism to ensure that ail kinetochores can reach the spindle equator. Xkid is thus a KLP involved in the movement of specific cargoes, chro­mosome arms, on microtubules. In contrast, Xklpl, another chro­mosome associated KLP, seems to be mainly involved in the organ­ization of microtubules around the chromatin to form the bipolar spindle. In conclusion, it is now clear that chromosomes play a very important role during mitosis. They modulate microtubule dynam­ics, thеу contribute to the organization of microtubules into a bipolar spindle and thеy control their own movements to align them on the metaphase plate.

Scholey addressed the role of forces produced by motors in spindle assembly and stability. Hе looked at spindle pole position in early drosophila embryos. They are 7-10 microns apart in metaphase and from 10 to 14 microns apart in anaphase. He pos­tulates that a balance of forces, produced by rotors, holds spindle poles in a steady state position. The motor KLP61F is involved in mitosis since loss of function gives abnormal spindles. KLP61F was purified from drosophila egg extracts and found to have a native mol. wt. of 550 kDa. In rotary shadowing it was found to be four-headed. It is localized in the nucleus and in spindles where it cross-links microtubules (homolog of Eg5). The injection of antibodies does not block separation at prometaphase, but induces a collapse at metaphase when the nucleus breaks down (collapse rate is 0.1 micron/sec). The minus end motor NCD is in the over­lap region. Thus, this suggests that NCD (dimer) opposes the plus end bipolar kinesin. And indeed, Ab to KLP61F did not cause spindle collapse in NCD mutant background (total null). This sug­gests that opposing forces on MTs by opposing rotors participate in the steady state length and organization of the spindle. In anaphase, NCD has no effect. CD inhibition blocks elongation. Blocking KLP61F blocks elongation. This suggests that CD and KLP61F both push poles apart. Scholey commented also on another topic dealing with raft transport in cilia and Kiensin 2. Using GFP-tagged proteins to follow raft motility, he showed that Kin II (КRP85 and 95 plus KR2115) drives raft transport. He put in KAP-GFP, OSM-6 and OSM-3-GFP chimeras into worms and anesthetized the worms. They move both anterograde and retro­grade directions at about 1 micron/sec. They looked in a Che-3 mutant background and found inhibition of transport retrogradely on cilia but not on dendrites.

Nedelec addressed the problem of microtubule-motor self­organization in a different way by using computer simulations and a simple in vitro system, composed just of tubulin and rotors. With mixtures of kinesin or NCD and MTs, asters form. Kinesin is streptavidin crosslinked and NCD antibody crosslinked. With kinesin, microtubule plus ends are in the center. With NCD, minus ends are in the middle.

With a mixture of tetrameric rotors, there is a separation of rotors in foc! that form quadrangles. In quads, there is a regular alternance Kin-NCD-Kin-NCD with microtubules having the same orientation between two piles.

Similar figures are reproduced in stochastic computer simula­tions. This model incorporates all the physical parameters associ­ated with rotors and microtubules and the computer calculates diffusion and stochastic interactions. All figures observed in exper­iments arise spontaneously in the computer over fairly broad parameter ranges. One parameter is important for the formation of asters: rotors should not fall off microtubule ends too fast. This new approach is very important to describe, in a predictive way, highly dynamic and complex systems.

Motors and Energy Production

The only speaker who addressed this question during this meet­ing was Kinoshita. It was interesting to discuss this aspect because the molecule he was talking about does the opposite to the rotors that had dominated the meeting: it does not use energy to produce movement; it generates energy (ATP) by moving – this is the F1/FO ATP synthase. Inner mitochondrial membrane uses electro­chemical gradient of protons to produce ATP. Recent work in Dimroth’s lab has indicated that proton gradient alone is not sufficient and transmembrane voltage is required for ATP synthesis.

The protein machine responsible for the synthеsis reaction is ATP synthase. This has been shown to be a rotary motor, second to the bacterial flagellar motor. So far, only two rotary molecular motors are known, compared to linear rotors for which a large variety exists. At least at the level of individuals, rotary mechanism is scarce or virtually absent. There is a reason for this: you need to have rel­ative displacement to get rotary motion. Thus, the rotor and stator must be separate entities, whereas a living organism is continuous. At the level of molecules, however, sliding is common and rotation is conceivable. For the ATP synthase, Boyer proposed some 20 years ago that it comprises two rotary rotors: F0 is in membrane and is a motor (turbine) driven by proton flow, whereas F1 is on the surface of the membrane and is a motor driven by ATP hydrol­yses. The two motors have a common rotor shaft, but the genuine rotary directions of the two are different: F0 tries to rive the shaft, say, clockwise, and F1 counterclockwise. In our body the proton motive force is large and thus F0 wins the game; F1 is forсibly rotated in its reverse direction, thus synthesizing ATP. In vitro, ATP hydrolysis in F1 can rotate the shaft in the opposite direction, resulting in pumping of protons. This Boyer model did not attract people until Walker and colleagues solved a Crystal struc­ture of F1. A gamma subunit was at the center and was surrounded by an orange shaped cylinder made of three alpha and three beta subunits. Catalytic sites were known to be on betas. Thus, the cylinder appeared as the driving stator and the central gamma the rotor. Tо prove this, an actin filament was attached to gamma through biotin-avidin-biotin link. Propeller rotation of the filament was seen under an optical microscope, showing that the gamma subunit truly rotates against the alpha3beta3 cylinder using ATP. Rotation was slowed by frictional load. A spec sheet of this motor contains the following: the motor is made of a single molecule (alpha3, beta3, and gamma subunits suffice). Rotation has been confirmed in TF 1, FF 1, and CF 1. In all cases, the direction is counter clockwise when viewed from F0 to F1. This is consistent with the clockwise arrangement of ATP-ADP-empty beta subunits in the Walker structure. TF1 motor rotates at 6000 rpm at room temperature, while its optimum temperature is aboye 50 degree C. 120° steps are resolved at 1ow ATP concentrations. Torque is 40 pNnm and is constant independent of load or speed. Work per 120° is about 80 pNnm, which is an efficiency of 80-100%. Input (free energy of ATP hydrolysis) can be reduced to 80 pNnm and the motor still rotate, suggesting that it can work with nearly 100% efficiency. Averaging many steps indicates that the speed of movement is constant, independent of position of shaft, and thus the torque is also constant independent of the shaft angle.

Model of movement

At very low ATP concentrations, F1 works in the uni-site mode where at most one beta subunit has nucleotide bound. At interme­diate concentrations, bi-site mode applies in which the binding of ATP to a second beta increases the release rate for ADP from the first site by more than 1000-fold, which would favor forward motion. Tri-site mode is suggested at high ATP concentrations, but Kinosita does not favor this idea.

Polarized fluorescence showed rotation of a fluorophore bound to the gamma subunit. Three polarization levels were observed, which is consistent with stepping at 120°. In another experiment, the fluorophore was excited with a linearly polarized light, of which the direction of polarization was rotated regularly. The phase of blinking of the fluorescence depends on the fluorophore orienta­tion. Again, 120° steps were resolved. In both experiments, the rate of ATP binding was estimated at about 3X 107 per M per sec. Experiments with actin also gave this value, implying that the rate of ATP binding is insensitive to the load.

Rotational speed measured with 93nm bead aggregates was at about 80 rps, and 120° stopping points were found, even at this speed. When 40 nm gold was observed in dark-field microscopy, rotation speed was 100 rps and each 120° stepping occurred in less than 0.1 msec.

ATP Production by Motor

The Walker crystal structure indicates that the beta subunit bend toward or off gamma, depending on whether the beta binds a nucleotide or not. This push-pull action, when properly coordi­nated, could produce rotation of gamma. This mechanism will require switches such that one side of gamma favors ATP binding and the other favors the release of ADP (Wang and Oster model). A switch-less model is also conceivable, as suggested by Oosawa and Hayashi.

Motors and sensor systems

Prost discussed a vеry original problem in terms of phуsical properties of a collection of molecules composing a sensory organ in the ear: the Hair Cells that Sense Sound. Hair cells emit sound, but fatigue occurs over the time scale of seconds. This can be explained as a phenomenon of self-tuned criticality. The hair сell bundle is coupled to upper basilar membrane and shear produced by the sound wave propagating in the surrounding medium is translated into a signal. Tip links are needed for the conversion of force into the opening of a calcium gate on adjacent bundles. The modeling of motor movement and oscillatory phenomena is neces­sary to understand this process. With a single motor, you have a сyсlic interaction between the motor and the periodic structure of the filament. With multiple motors, уou have multiple interactions, which could give rise to a dynamiсal phase transition. One can get regions of negative velocity at high forces. Adding an elastic element to the system (like titin) will give rise to an oscillator. The frequency of the oscillations will be related to the motor frequency but can be larger. The oscillation frequency is such that the dissi­pative losses are balanced by the motors work, and two character­istic frequencies come into play: the motors transition rate, and the inverse response time of the elastic element. The obtained fre­quency is the harmonic mean between the two, which can be larger than the motor cycle. Fly and bee wings oscillate at frequencies greater than the speed of contraction and relaxation. Ciliary Beating Characteristic frequency was predicted from the reasonable elastic and force numbers for the filament. The frequency is pro­portional to motor frequency, stiffness constant and the inverse of square of cilium length and viscosity of the medium. Gain of the system is proportional to the inverse of the force to the 2/3 power. Noise in the system will not be in phase but signal will have phase coherence. Hearing response goes as the 1/3 of the power for inter­mediate power, but then rises linearly at the highest intensities. Frequency coherence gives rise to response that is cleaner because of reinforcement. This is a Tortini effect, in other words a harmonie sensing. Fatigue arises from calcium concentration rise in the cell and the need for pumps to catch up.


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