Comprehensive comparative analysis of kinesins in photosynthetic eukaryotes

Background

Kinesins, a superfamily of molecular motors, use microtubules as tracks and transport diverse cellular cargoes. All kinesins contain a highly conserved ~350 amino acid motor domain. Previous analysis of the completed genome sequence of one flowering plant (Arabidopsis) has resulted in identification of 61 kinesins. The recent completion of genome sequencing of several photosynthetic and non-photosynthetic eukaryotes that belong to divergent lineages offers a unique opportunity to conduct a comprehensive comparative analysis of kinesins in plant and non-plant systems and infer their evolutionary relationships.

Results

We used the kinesin motor domain to identify kinesins in the completed genome sequences of 19 species, including 13 newly sequenced genomes. Among the newly analyzed genomes, six represent photosynthetic eukaryotes. A total of 529 kinesins was used to perform comprehensive analysis of kinesins and to construct gene trees using the Bayesian and parsimony approaches. The previously recognized 14 families of kinesins are resolved as distinct lineages in our inferred gene tree. At least three of the 14 kinesin families are not represented in flowering plants. Chlamydomonas, a green alga that is part of the lineage that includes land plants, has at least nine of the 14 known kinesin families. Seven of ten families present in flowering plants are represented in Chlamydomonas, indicating that these families were retained in both the flowering-plant and green algae lineages.

Conclusion

The increase in the number of kinesins in flowering plants is due to vast expansion of the Kinesin-14 and Kinesin-7 families. The Kinesin-14 family, which typically contains a C-terminal motor, has many plant kinesins that have the motor domain at the N terminus, in the middle, or the C terminus. Several domains in kinesins are present exclusively either in plant or animal lineages. Addition of novel domains to kinesins in lineage-specific groups contributed to the functional diversification of kinesins. Results from our gene-tree analyses indicate that there was tremendous lineage-specific duplication and diversification of kinesins in eukaryotes. Since the functions of only a few plant kinesins are reported in the literature, this comprehensive comparative analysis will be useful in designing functional studies with photosynthetic eukaryotes.     

Kinesin tail domains are intrinsically disordered

Kinesin motor proteins transport a wide variety of molecular cargoes in a spatially and temporally regulated manner. Kinesin motor domains, which hydrolyze ATP to produce a directed mechanical force along a microtubule, are well conserved throughout the entire superfamily. Outside of the motor domains, kinesin sequences diverge along with their transport functions. The nonmotor regions, particularly the tails, respond to a wide variety of structural and molecular cues that enable kinesins to carry specific cargoes in response to particular cellular signals. Here, we demonstrate that intrinsic disorder is a common structural feature of kinesins. A bioinformatics survey of the full‐length sequences of all 43 human kinesins predicts that significant regions of intrinsically disordered residues are present in all kinesins. These regions are concentrated in the nonmotor domains, particularly in the tails and near sites for ligand binding or post‐translational modifications. In order to experimentally verify these predictions, we expressed and purified the tail domains of kinesins representing three different families (Kif5B, Kif10, and KifC3). Circular dichroism and NMR spectroscopy experiments demonstrate that the isolated tails are disordered in vitro, yet they retain their functional microtubule‐binding activity. On the basis of these results, we propose that intrinsic disorder is a common structural feature that confers functional specificity to kinesins. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

Maximum Likelihood Methods Reveal Conservation of Function Among Closely Related Kinesin Families

We have reconstructed the evolution of the anciently derived kinesin superfamily using various alignment and tree-building methods. In addition to classifying previously described kinesins from protists, fungi, and animals, we analyzed a variety of kinesin sequences from the plant kingdom including 12 from Zea mays and 29 from Arabidopsis thaliana. Also included in our data set were four sequences from the anciently diverged amitochondriate protist Giardia lamblia. The overall topology of the best tree we found is more likely than previously reported topologies and allows us to make the following new observations: (1) kinesins involved in chromosome movement including MCAK, chromokinesin, and CENP-E may be descended from a single ancestor; (2) kinesins that form complex oligomers are limited to a monophyletic group of families; (3) kinesins that crosslink antiparallel microtubules at the spindle midzone including BIMC, MKLP, and CENP-E are closely related; (4) Drosophila NOD and human KID group with other characterized chromokinesins; and (5) Saccharomyces SMY1 groups with kinesin-I sequences, forming a family of kinesins capable of class V myosin interactions. In addition, we found that one monophyletic clade composed exclusively of sequences with a C-terminal motor domain contains all known minus end-directed kinesins. Received: 20 February 2001 / Accepted: 5 June 2001     

Functions of the Arabidopsis kinesin superfamily of microtubule-based motor proteins

Plants possess a large number of microtubule-based kinesin motor proteins. While the kinesin-2, 3, 9, and 11 families are absent from land plants, the kinesin-7 and 14 families are greatly expanded. In addition, some kinesins are specifically present only in land plants. The distinctive inventory of plant kinesins suggests that kinesins have evolved to perform specialized functions in plants. Plants assemble unique microtubule arrays during their cell cycle, including the interphase cortical microtubule array, preprophase band, anastral spindle and phragmoplast. In this review, we explore the functions of plant kinesins from a microtubule array viewpoint, focusing mainly on Arabidopsis kinesins. We emphasize the conserved and novel functions of plant kinesins in the organization and function of the different microtubule arrays.     

One axon,many kinesins: What's the logic?

A large number of membrane-bounded organelles, protein complexes, and mRNAs are transported along microtubules to different locations within the neuronal axon. Axonal transport in the anterograde direction is carried out by members of a superfamily of specialized motor proteins, the kinesins. All kinesins contain a conserved motor domain that hydrolyses ATP to generate movement along microtubules. Regions outside the motor domain are responsible for cargo binding and regulation of motor activity. Present in a soluble, inactive form in the cytoplasm, kinesins are activated upon cargo binding. Selective targeting of different types of kinesin motors to specific cargoes is directed by amino acid sequences situated in their variable tails. Cargo proteins with specific function at their destination, bind directly to specific kinesins for transport. Whereas most kinesins move to microtubule plus-ends, a small number of them move to microtubule minus-ends, and may participate in retrograde axonal transport. Axonal transport by kinesins has a logic: Fully assembled, multisubunit, functional complexes (e.g., ion channel complexes, signaling complexes, RNA-protein complexes) are transported to their destination by kinesin motors that interact transiently (i.e., during transport only) with one of the complexes' subunits.     

Essential kinesins: characterization of Caenorhabditis elegans KLP-15

Kinesins form a superfamily of molecular motors involved in cell division and intracellular transport. Twenty kinesins have been found in the Caenorhabditis elegans genome, and four of these belong to the kinesin-14 subfamily, i.e., kinesins with C-terminal motor domains. Three of these kinesin-14s, KLP-15, KLP-16, and KLP-17, form a distinct subgroup in which KLP-15 and KLP-16 are more than 90% identical and appear to be related by a relatively recent gene duplication. They are essential for meiotic spindle organization and chromosome segregation, and are mostly expressed in the germline. With 587 amino acids each, they are among the smallest kinesins known. Using bacterially expressed KLP-15 constructs with different length extensions preceding the motor domain, we have determined in vitro the following characteristic properties: ATPase activity, microtubule binding, oligomeric state, microtubule gliding activity, and direction of movement. The constructs exhibit a monomer-dimer equilibrium that depends on the length of the predicted alpha-helical coiled-coil region preceding the motor domain. The longest construct with the complete coiled-coil domain is a stable dimer, and the shortest construct with only seven amino acids preceding the motor domain is a monomer. In microtubule gliding assays, the monomer is immobile whereas the fully dimeric KLP-15 construct supports gliding at 2.3 microm/min and moves toward microtubule minus ends, like other members of the kinesin-14 subfamily studied to date.     

Functional analysis of human microtubule-based motor proteins, the kinesins and dyneins, in mitosis/cytokinesis using RNA interference

Microtubule (MT)-based motor proteins, kinesins and dyneins, play important roles in multiple cellular processes including cell division. In this study, we describe the generation and use of an Escherichia coli RNase III-prepared human kinesin/dynein esiRNA library to systematically analyze the functions of all human kinesin/dynein MT motor proteins. Our results indicate that at least 12 kinesins are involved in mitosis and cytokinesis. Eg5 (a member of the kinesin-5 family), Kif2A (a member of the kinesin-13 family), and KifC1 (a member of the kinesin-14 family) are crucial for spindle formation; KifC1, MCAK (a member of the kinesin-13 family), CENP-E (a member of the kinesin-7 family), Kif14 (a member of the kinesin-3 family), Kif18 (a member of the kinesin-8 family), and Kid (a member of the kinesin-10 family) are required for chromosome congression and alignment; Kif4A and Kif4B (members of the kinesin-4 family) have roles in anaphase spindle dynamics; and Kif4A, Kif4B, MKLP1, and MKLP2 (members of the kinesin-6 family) are essential for cytokinesis. Using immunofluorescence analysis, time-lapse microscopy, and rescue experiments, we investigate the roles of these 12 kinesins in detail.     

Universal and unique features of kinesin motors: insights from a comparison of fungal and animal conventional kinesins.

Kinesins are microtubule motors that use the energy derived from the hydrolysis of ATP to move unidirectionally along microtubules. The founding member of this still growing superfamily is conventional kinesin, a dimeric motor that moves processively towards the plus end of microtubules. Within the family of conventional kinesins, two groups can be distinguished to date, one derived from animal species, and one originating from filamentous fungi. So far no conventional kinesin has been reported from plant cells. Fungal and animal conventional kinesins differ in several respects, both in terms of their primary sequence and their physiological properties. Thus all fungal conventional kinesins move at velocities that are 4-5 times higher than those of animal conventional kinesins, and all of them appear to lack associated light chains. Both groups of motors are characterized by a number of group-specific sequence features which are considered here with respect to their functional importance. Animal and fungal conventional kinesins also share a number of sequence characteristics which point to common principles of motor function. The overall domain organization is remarkably similar. A C-terminal sequence motif common to all kinesins, which constitutes the only region of high homology outside the motor domain, suggests common principles of cargo association in both groups of motors. Consideration of the differences of, and similarities between, fungal and animal kinesins offers novel possibilities for experimentation (e. g., by constructing chimeras) that can be expected to contribute to our understanding of motor function.     

KIF14 Binds Tightly to Microtubules and Adopts a Rigor-Like Conformation

The mitotic kinesin motor protein KIF14 is essential for cytokinesis during cell division and has been implicated in cerebral development and a variety of human cancers. Here we show that the mouse KIF14 motor domain binds tightly to microtubules and does not display typical nucleotide-dependent changes in this affinity. It also has robust ATPase activity but very slow motility. A crystal structure of the ADP-bound form of the KIF14 motor domain reveals a dramatically opened ATP-binding pocket, as if ready to exchange its bound ADP for Mg·ATP. In this state, the central β-sheet is twisted ~ 10° beyond the maximal amount observed in other kinesins. This configuration has only been seen in the nucleotide-free states of myosins—known as the “rigor-like” state. Fitting of this atomic model to electron density maps from cryo-electron microscopy indicates a distinct binding configuration of the motor domain to microtubules. We postulate that these properties of KIF14 are well suited for stabilizing midbody microtubules during cytokinesis.     

A mechanism for microtubule depolymerization by KinI kinesins

Whereas most kinesins motor along microtubules, KinI kinesins are microtubule depolymerizing machines. Surprisingly, we found that a KinI fragment consisting of only the motor core is capable of ATP-dependent depolymerization. The motor binds along microtubules in all nucleotide states, but in the presence of AMPPNP, microtubule depolymerization also occurs. Structural characterization of the products of AMPPNP-induced destabilization revealed a snapshot of the disassembly machine in action as it precisely deformed a tubulin dimer. While conventional kinesins use the energy of ATP binding to execute a "powerstroke," KinIs use it to bend the underlying protofilament. Thus, the relatively small class-specific differences within the KinI motor core modulate a fundamentally conserved mode of interaction with microtubules to produce a unique depolymerizing activity.     

How kinesin motor proteins drive mitotic spindle function: Lessons from molecular assays

Kinesins are enzymes that use the energy of ATP to perform mechanical work. There are approximately 14 families of kinesins within the kinesin superfamily. Family classification is derived primarily from alignments of the sequences of the core motor domain. For this reason, the enzymatic behavior and motility of each motor generally reflects its family. At the cellular level, kinesin motors perform a variety of functions during cell division and within the mitotic spindle to ensure that chromosomes are segregated with the highest fidelity possible. The cellular functions of these motors are intimately related to their mechanical and enzymatic properties at the single molecule level. For this reason, motility studies designed to evaluate the activity of purified molecular motors are a requirement in order to understand, mechanistically, how these motors make the mitotic spindle work and what can cause the spindle to fail. This review will focus on a selection of illustrative kinesins, which have been studied at the molecular level in order to inform our understanding of their function in cells. In addition, the review will endeavor to point out some kinesins that have been studied extensively but which still lack sufficient molecular underpinnings to fully predict their contribution to spindle function.     

Unconventional motoring: an overview of the Kin C and Kin I kinesins

All kinesins share a conserved core motor domain implying a common mechanism for generating force from ATP hydrolysis. How is it then that kinesins exhibit such divergent activities: motility, microtubule cross‐linking and microtubule depolymerization? Although conventional motile kinesins have served as the paradigm for understanding kinesin function, the unconventional kinesins exploit variations on the motile theme to perform unexpected tasks. This review summarizes the biological functions and examines the possible molecular mechanisms of Kin C and Kin I unconventional kinesins. We also discuss the possible differences between the microtubule destabilization models proposed for Kar3 and Kin I kinesins .     

Motoring down the highways of the cell

All eukaryotic cells contain large numbers of motor proteins (kinesins, dyneins and myosins), each of which appears to carry out a specialized force-generating function within the cell. They are known to have roles in muscle contraction, ciliary movement, organelle and vesicle transport, mitosis and cytokinesis. These motor proteins operate on different cytoskeletal filaments; myosins move along actin filaments, and kinesins and dyneins along microtubules. Recently published crystal structures of the motor domains of two members of the kinesin superfamily reveal that they share the same overall fold that is also found at the core of the larger myosin motor. This suggests that they may share a common mechanism as well as a common ancestry.     

Metazoan motor models: kinesin superfamily in C. elegans

In eukaryotic cells members of the kinesin family mediate intracellular transport by carrying cellular cargo on microtubule tracks. The nematode Caenorhabditis elegans genome encodes 21 members of the kinesin family, which show significant homology to their mammalian orthologs. Based on motor domain sequence homology and placement of the motor domain in the protein, the C. elegans kinesins have been placed in eight distinct groups; members of which participate in embryonic development, protein transport, synaptic membrane vesicles movement and in the axonal growth. Among 21 kinesins, at least 11 play a central role in spindle movement and chromosomal segregation. Understanding the function of C. elegans kinesins and related proteins may help navigate through the intricacies of intracellular traffic in a simple animal.     

The kinesin-13 MCAK has an unconventional ATPase cycle adapted for microtubule depolymerization

Unlike other kinesins, members of the kinesin-13 subfamily do not move directionally along microtubules but, instead, depolymerize them. To understand how kinesins with structurally similar motor domains can have such dissimilar functions, we elucidated the ATP turnover cycle of the kinesin-13, MCAK. In contrast to translocating kinesins, ATP cleavage, rather than product release, is the rate-limiting step for ATP turnover by MCAK; unpolymerized tubulin and microtubules accelerate this step. Further, microtubule ends fully activate the ATPase by accelerating the exchange of ADP for ATP. This tuning of the cycle adapts MCAK for its depolymerization activity: lattice-stimulated ATP cleavage drives MCAK into a weakly bound nucleotide state that reaches microtubule ends by diffusion, and end-specific acceleration of nucleotide exchange drives MCAK into a strongly bound state that promotes depolymerization. This altered cycle accounts well for the different mechanical behaviour of this kinesin, which depolymerizes microtubules from their ends, compared to translocating kinesins that walk along microtubules. Thus, the kinesin motor domain is a nucleotide-dependent engine that can be differentially tuned for transport or depolymerization functions.     

Directionality of kinesin motors

Kinesins are molecular motors that transport various cargoes along microtubule tracks using energy derived from ATP hydrolysis. Although the motor domains of kinesins are structurally similar, the family contains members that move on microtubules in opposite directions. Recent biochemical and biophysical studies of several kinesins make it possible to identify structural elements responsible for the different directionality, suggesting that reversal of the motor movement can be achieved through small, local changes in the protein structure.     

Identification and phylogenetic analysis ofDictyostelium discoideumkinesin proteins

Background

Kinesins constitute a large superfamily of motor proteins in eukaryotic cells. They perform diverse tasks such as vesicle and organelle transport and chromosomal segregation in a microtubule- and ATP-dependent manner. In recent years, the genomes of a number of eukaryotic organisms have been completely sequenced. Subsequent studies revealed and classified the full set of members of the kinesin superfamily expressed by these organisms. ForDictyostelium discoideum, only five kinesin superfamily proteins (Kif's) have already been reported.

Results

Here, we report the identification of thirteen kinesin genes exploiting the information from the raw shotgun reads of theDictyostelium discoideumgenome project. A phylogenetic tree of 390 kinesin motor domain sequences was built, grouping theDictyosteliumkinesins into nine subfamilies. According to known cellular functions or strong homologies to kinesins of other organisms, four of theDictyosteliumkinesins are involved in organelle transport, six are implicated in cell division processes, two are predicted to perform multiple functions, and one kinesin may be the founder of a new subclass.

Conclusion

This analysis of theDictyosteliumgenome led to the identification of eight new kinesin motor proteins. According to an exhaustive phylogenetic comparison,Dictyosteliumcontains the same subset of kinesins that higher eukaryotes need to perform mitosis. Some of the kinesins are implicated in intracellular traffic and a small number have unpredictable functions.     

An internal motor kinesin is associated with the Golgi apparatus and plays a role in trichome morphogenesis in Arabidopsis

Members of the kinesin superfamily are microtubule-based motor proteins that transport molecules/organelles along microtubules. We have identified similar internal motor kinesins, Kinesin-13A, from the cotton Gossypium hirsutum and Arabidopsis thaliana. Their motor domains share high degree of similarity with those of internal motor kinesins of animals and protists in the MCAK/Kinesin13 subfamily. However, no significant sequence similarities were detected in sequences outside the motor domain. In Arabidopsis plants carrying the T-DNA knockout kinesin-13a-1 and kinesin-13a-2 mutations at the Kinesin-13A locus, >70% leaf trichomes had four branches, whereas wild-type trichomes had three. Immunofluorescent results showed that AtKinesin-13A and GhKinesin-13A localized to entire Golgi stacks. In both wild-type and kinesin-13a mutant cells, the Golgi stacks were frequently associated with microtubules and with actin microfilaments. Aggregation/clustering of Golgi stacks was often observed in the kinesin-13a mutant trichomes and other epidermal cells. This suggested that the distribution of the Golgi apparatus in cell cortex might require microtubules and Kinesin-13A, and the organization of Golgi stacks could play a regulatory role in trichome morphogenesis. Our results also indicate that plant kinesins in the MCAK/Kinesin-13 subfamily have evolved to take on different tasks than their animal counterparts.