Role of Unc104/KIF1-related motor proteins in mitochondrial transport in Neurospora crassa

Eukaryotic cells use diverse cytoskeleton-dependent machineries to control inheritance and intracellular positioning of mitochondria. In particular, microtubules play a major role in mitochondrial motility in the filamentous fungus Neurospora crassa and in mammalian cells. We examined the role of two novel Unc104/KIF1-related members of the kinesin family, Nkin2 and Nkin3, in mitochondrial motility in Neurospora. The Nkin2 protein is required for mitochondrial interactions with microtubules in vitro. Mutant hyphae lacking Nkin2 show mitochondrial motility defects in vivo early after germination of conidiospores. Nkin3, a member of a unique fungal-specific subgroup of small Unc104/KIF1-related proteins, is not associated with mitochondria in wild-type cells. However, it is highly expressed and recruited to mitochondria in Deltankin-2 mutants. Mitochondria lacking Nkin2 require Nkin3 for binding to microtubules in vitro, and mitochondrial motility defects in Deltankin-2 mutants disappear with up-regulation of Nkin3 in vivo. We propose that mitochondrial transport is mediated by Nkin2 in Neurospora, and organelle motility defects in Deltankin-2 mutants are rescued by Nkin3. Apparently, a highly versatile complement of organelle motors allows the cell to efficiently respond to exogenous challenges, a process that might also account for the great variety of different mitochondrial transport systems that have evolved in eukaryotic cells.     

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.     

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.     

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.     

Structural comparison of dimeric Eg5, Neurospora kinesin (Nkin) and Ncd head-Nkin neck chimera with conventional kinesin

Cryo-electron microscopy and 3D image reconstruction of microtubules saturated with kinesin dimers has shown one head bound to tubulin, the other free. The free head of rat kinesin sits on the top right of the bound head (with the microtubule oriented plus-end upwards) in the presence of 5'-adenylylimido-diphosphate (AMPPNP) and on the top left in nucleotide-free solutions. To understand the relevance of this movement, we investigated other dimeric plus-end-directed motors: Neurospora kinesin (Nkin); Eg5, a slow non-processive kinesin; and a chimera of Ncd heads attached to Nkin necks. In the AMPPNP (ATP-like) state, all dimers have the free head to the top right. In the absence of nucleotide, the free head of an Nkin dimer appears to occupy alternative positions to either side of the bound head. Despite having the Nkin neck, the free head of the chimera was only seen to the top right of the bound head. Eg5 also has the free head mostly to the top right. We suggest that processive movement may require kinesins to move their heads in alternative ways.     

The family-specific K-loop influences the microtubule on-rate but not the superprocessivity of kinesin-3 motors

The kinesin-3 family (KIF) is one of the largest among the kinesin superfamily and an important driver of a variety of cellular transport events. Whereas all kinesins contain the highly conserved kinesin motor domain, different families have evolved unique motor features that enable different mechanical and functional outputs. A defining feature of kinesin-3 motors is the presence of a positively charged insert, the K-loop, in loop 12 of their motor domains. However, the mechanical and functional output of the K-loop with respect to processive motility of dimeric kinesin-3 motors is unknown. We find that, surprisingly, the K-loop plays no role in generating the superprocessive motion of dimeric kinesin-3 motors (KIF1, KIF13, and KIF16). Instead, we find that the K-loop provides kinesin-3 motors with a high microtubule affinity in the motor''s ADP-bound state, a state that for other kinesins binds only weakly to the microtubule surface. A high microtubule affinity results in a high landing rate of processive kinesin-3 motors on the microtubule surface. We propose that the family-specific K-loop contributes to efficient kinesin-3 cargo transport by enhancing the initial interaction of dimeric motors with the microtubule track.     

Properties of the Kinesin-1 motor DdKif3 from Dictyostelium discoideum

The amoeba Dictyostelium discoideum possesses genes for 13 different kinesins. Here we characterize DdKif3, a member of the Kinesin-1 family. Kinesin-1 motors form homodimers that can move micrometer-long distances on microtubules using the energy derived from ATP hydrolysis. We expressed recombinant motors in Escherichia coli and tested them in different in vitro assays. Full-length and truncated Kif3 motors were active in gliding and ATPase assays. They showed a strong dependence on ionic strength. Like the full-length motor, the truncated DdKif3-592 motor (aa 1-592; comprising motor domain, neck, and partial stalk) reached its maximum speed of around 2.0micrcom s(-1) at a potassium acetate concentration of 200mM. The shortened DdKif3-342 motor (aa 1-342; comprising motor domain, partial neck) showed a high ATP turnover, comparable to that of the fungal Kinesin-1, Nkin. Results from the duty cycle calculations and gliding assays indicate that DdKif3 is a processive motor. A GFP-fusion protein revealed a mainly cytoplasmic localization of DdKif3. Immunofluorescence staining makes an association with the endoplasmic reticulum or mitochondria unlikely. Despite a similar phylogenetic distance to both metazoa and fungi, in terms of its biochemical properties DdKif3 revealed a closer similarity to fungal than animal kinesins.     

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.     

Kinesin: walking, crawling or sliding along?

Kinesins are microtubule-based motor proteins that are involved in cargo transport and mitosis. They are called "motors" because they convert chemical energy to mechanical energy (i.e. force and motion). They use the energy of ATP hydrolysis for their enzymatic processes by walking on microtubules. However, the mechanism underlying their motion has been unclear. Recently, conventional kinesin, which was the first-identified member of the family, has been shown to walk by swapping its two heads in a "hand-over-hand" mechanism. There is also experimental evidence supporting an asymmetric walking of kinesin in which two identical heads of the motor take alternate slow and fast steps. Other cargo-carrier and mitotic kinesins remain uninvestigated and are of great interest to biophysicists.     

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.     

Kinesin proteins: A phylum of motors for microtubule-based motility

The cellular processes of transport, division and, possibly, early development all involve microtubule-based motors. Recent work shows that, unexpectedly, many of these cellular functions are carried out by different types of kinesin and kinesin-related motor proteins. The kinesin proteins are a large and rapidly growing family of microtubule-motor proteins that share a 340-amino-acid motor domain. Phylogenetic analysis of the conserved motor domains groups the kinesin proteins into a number of subfamilies, the members of which exhibit a common molecular organization and related functions. The kinesin proteins that belong to different subfamilies differ in their rates and polarity of movement along microtubules, and probably in the particles/organelles that they transport. The kinesins arose early in eukaryotic evolution and gene duplication has allowed functional specialization to occur, resulting in a surprisingly large number of different classes of these proteins adapted for intracellular transport of vesicles and organelles, and for assembly and force generation in the meiotic and mitotic spindles.     

A novel split kinesin assay identifies motor proteins that interact with distinct vesicle populations

Identifying the kinesin motors that interact with different vesicle populations is a longstanding and challenging problem with implications for many aspects of cell biology. Here we introduce a new live-cell assay to assess kinesin-vesicle interactions and use it to identify kinesins that bind to vesicles undergoing dendrite-selective transport in cultured hippocampal neurons. We prepared a library of "split kinesins," comprising an axon-selective kinesin motor domain and a series of kinesin tail domains that can attach to their native vesicles; when the split kinesins were assembled by chemical dimerization, bound vesicles were misdirected into the axon. This method provided highly specific results, showing that three Kinesin-3 family members-KIF1A, KIF13A, and KIF13B-interacted with dendritic vesicle populations. This experimental paradigm allows a systematic approach to evaluate motor-vesicle interactions in living cells.     

Importance of a flexible hinge near the motor domain in kinesin-driven motility.

Conventional kinesin is a molecular motor consisting of an N-terminal catalytic motor domain, an extended stalk and a small globular C-terminus. Whereas the structure and function of the catalytic motor domain has been investigated, little is known about the function of domains outside the globular head. A short coiled-coil region adjacent to the motor domain, termed the neck, is known to be important for dimerization and may be required for kinesin processivity. We now provide evidence that a helix-disrupting hinge region (hinge 1) that separates the neck from the first extended coiled-coil of the stalk plays an essential role in basic motor activity. A fast fungal kinesin from Syncephalastrum racemosum was used for these studies. Deletion, substitution by a coiled-coil and truncation of the hinge 1 region all reduce motor speed and uncouple ATP turnover from gliding velocity. Insertion of hinge 1 regions from two conventional kinesins, Nkin and DmKHC, fully restores motor activity, whereas insertion of putative flexible linkers of other proteins does not, suggesting that hinge 1 regions of conventional kinesins can functionally replace each other. We suggest that this region is essential for kinesin movement in its promotion of chemo-mechanical coupling of the two heads and therefore the functional motor domain should be redefined to include not only the catalytic head but also the adjacent neck and hinge 1 domains.     

Single fungal kinesin motor molecules move processively along microtubules

Conventional kinesins are two-headed molecular motors that move as single molecules micrometer-long distances on microtubules by using energy derived from ATP hydrolysis. The presence of two heads is a prerequisite for this processive motility, but other interacting domains, like the neck and K-loop, influence the processivity and are implicated in allowing some single-headed kinesins to move processively. Neurospora kinesin (NKin) is a phylogenetically distant, dimeric kinesin from Neurospora crassa with high gliding speed and an unusual neck domain. We quantified the processivity of NKin and compared it to human kinesin, HKin, using gliding and fluorescence-based processivity assays. Our data show that NKin is a processive motor. Single NKin molecules translocated microtubules in gliding assays on average 2.14 micro m (N = 46). When we tracked single, fluorescently labeled NKin motors, they moved on average 1.75 micro m (N = 182) before detaching from the microtubule, whereas HKin motors moved shorter distances (0.83 micro m, N = 229) under identical conditions. NKin is therefore at least twice as processive as HKin. These studies, together with biochemical work, provide a basis for experiments to dissect the molecular mechanisms of processive movement.     

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.     

Diffusion of kinesin motors on cargo can enhance binding and run lengths during intracellular transport

Cellular cargoes, including lipid droplets and mitochondria, are transported along microtubules using molecular motors such as kinesins. Many experimental and computational studies focused on cargoes with rigidly attached motors, in contrast to many biological cargoes that have lipid surfaces that may allow surface mobility of motors. We extend a mechanochemical three-dimensional computational model by adding coupled-viscosity effects to compare different motor arrangements and mobilities. We show that organizational changes can optimize for different objectives: Cargoes with clustered motors are transported efficiently but are slow to bind to microtubules, whereas those with motors dispersed rigidly on their surface bind microtubules quickly but are transported inefficiently. Finally, cargoes with freely diffusing motors have both fast binding and efficient transport, although less efficient than clustered motors. These results suggest that experimentally observed changes in motor organization may be a control point for transport.     

The Translocation Selectivity of the Kinesins that Mediate Neuronal Organelle Transport

Polarized kinesin‐driven transport is crucial for development and maintenance of neuronal polarity. Kinesins are thought to recognize biochemical differences between axonal and dendritic microtubules in order to deliver their cargoes to the appropriate domain. To identify kinesins that mediate polarized transport, we prepared constitutively active versions of all the kinesins implicated in vesicle transport and expressed them in cultured hippocampal neurons. Seven kinesins translocated preferentially to axons and five translocated into both axons and dendrites. None translocated selectively to dendrites. Highly homologous members of the same subfamily displayed distinctly different translocation preferences and were differentially regulated during development. By expressing chimeric kinesins, we identified two microtubule‐binding elements within the motor domain that are important for selective translocation. We also discovered elements in the dimerization domain of kinesin‐2 motors that contribute to their selective translocation. These observations indicate that selective interactions between kinesin motor domains and microtubules can account for polarized transport to the axon, but not for selective dendritic transport.     

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 .     

Intrinsic disorder in the kinesin superfamily

Kinesin molecular motors perform a myriad of intracellular transport functions. While their mechanochemical mechanisms are well understood and well-conserved throughout the superfamily, the cargo-binding and regulatory mechanisms governing the activity of kinesins are highly diverse and, in general, incompletely characterized. Here we present evidence from bioinformatic predictions indicating that most kinesin superfamily members contain significant regions of intrinsically disordered (ID) residues. ID regions can bind to multiple partners with high specificity and are highly labile to post-translational modification and degradation signals. In kinesins, the predicted ID regions are primarily found in areas outside the motor domains, where primary sequences diverge by family, suggesting that the ID may be a critical structural element for determining the functional specificity of individual kinesins. To support this concept, we present a systematic analysis of the kinesin superfamily, family by family, for predicted ID regions. We combine this analysis with a comprehensive review of kinesin-binding partners and post-translational modifications. We find two key trends across the entire kinesin superfamily. First, ID residues tend to be in the tail regions of kinesins, opposite the superfamily-conserved motor domains. Second, predicted ID regions correlate to regions that are known to bind to cargoes and/or undergo post-translational modifications. We therefore propose that the ID residue is a structural element utilized by the kinesin superfamily in order to impart functional specificity to individual kinesins.     

A kinesin medley: biochemical and functional heterogeneity

Over the past decade, a remarkable number and diversity of molecular motors have been described in eukaryotic cells. In addition to the identification of novel forms of myosin and dynein, the kinesins have been defined as an entirely new family of molecular motors. There may be as many as 30 different genes in a single organism encoding members of the kinesin superfamily. Why is such diversity in molecular motors needed? The biochemical and functional diversity of the originally defined form of kinesin provides some insights into the roles of molecular motors in cellular dynamics.