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A “molecular guillotine” reveals an interphase function of Kinesin-5

Motor proteins are important for transport and force generation in a variety of cellular processes and morphogenesis. Here we design a general strategy for conditional motor mutants by inserting a protease cleavage site at the “neck” between the head domain and the stalk of the motor protein, making the protein susceptible to proteolytic cleavage at the neck by the corresponding protease. To demonstrate the feasibility of this approach, we inserted the cleavage site of TEV protease into the neck of the tetrameric motor Kinesin-5. Application of TEV protease led to a specific depletion and functional loss of Kinesin-5 in Drosophila embryos. By this, we revealed that Kinesin-5 stabilized the microtubule network during interphase in syncytial embryos. The “molecular guillotine” can potentially be applied to many motor proteins due to the conserved structures of kinesin, dynein and myosin with accessible necks. Author summary We design a general strategy for conditional motor mutants by inserting a protease cleavage site between head and stalk domain of the motor protein, making it susceptible to specific proteolytic cleavage. We demonstrate the feasibility of the approach with the motor Kinesin-5 and the protease TEV in Drosophila embryos. This approach can potentially be applied to motor proteins kinesin, dynein and myosin due to the conserved structures.


Introduction 8
Cytoskeletal motor proteins, including myosins, dyneins and kinesins, convert 9 the chemical energy of ATP hydrolysis into mechanical work. Motor proteins 10 are wildly involved in multiple fundamental cellular processes such as 11 intracellular transport, cell division, cell shape change and migration [1]. The 12 structure of motor proteins is conserved. They contain a motor domain, 13 referred to as "head", which catalyzes ATP and binds microtubules or F-actin. 14 The catalytic cycle links ATP hydrolysis to a conformational change of the 15 protein that translates into unidirectional movement of the motor protein on the 16 filament. A second part of the protein, the stalk, links the head to the cargo 17 binding site, contains coil-coiled structures for oligomerization or associates 18 with other subunits. Head and stalk are parts of the same polypeptide, which is 19 functionally relevant as a tight link of head and stalk is essential for 20 transmission of mechanical force [2]. 21 22 Genetic analysis of the physiological function of motor proteins is hampered, 23 since many motor proteins fulfill an essential function for the cell or organism. 24 For example, Kinesin-5 serves indispensable functions during mitosis, making 25 an analysis of its function in interphase or in terminally differentiated cells 26 difficult. Conditional mutations, such as temperature sensitive alleles, can 27 overcome these limits of genetic analysis [3]. Gene knock down by RNAi 28 approaches relays on protein turnover, leading to insensitivity of stable 1 proteins. Pharmacological approaches with small molecules inhibitors or 2 specific antibodies provide an alternative and have been applied for motor 3 protein inhibition [4][5][6]. However, chemical approaches cannot be generalized, 4 and need to be developed case by case. 5 6 Kinesin-5 belongs to kinesin family member 11 (KIF11), with the motor domain 7 on N terminus, followed by a coiled-coil rod containing a central bipolar 8 assembly (BASS) domain. Forming bipolar homo tetramers, Kinesin-5 can 9 crosslink anti-parallel aligned microtubules. The motor activity enables filament 10 sliding, e. g. during formation and elongation of the mitotic spindle [7]. In 11 Drosophila syncytial embryos, Kinesin-5 is enriched at mitotic spindles and is 12 essential for spindle formation and chromosome segregation. Injection of 13 antibodies specific for Kinesin-5 into embryos leads to collapse of newly 14 formed spindle and the formation of mono-asters of microtubules [5,6]. 15

16
Making proteins susceptible to proteolytic cleavage represents a generally 17 applicable strategy for generation of conditional alleles [8][9][10]. 18 Here we apply this concept to motor proteins by inserting a proteolytic site 19 between the head and stalk region ("neck"). We designated this strategy a 20 "molecular guillotine" (Fig. 1A). We chose well-characterized Kinesin-5 in order 21 to demonstrate the feasibility of this approach. As a protease, we employ TEV, 22 which is highly specific. No match of TEV recognition motif within the 23 Drosophila proteome has been identified, and flies expressing TEV are viable 24 and fertile [10]. 25 26

27
Design of a "molecular guillotine" 28 We inserted three copies of the TEV recognition motif at one of two positions, 1 G394 or Q499, into the stalk region. G394 and Q499 are located within 2 conserved coiled-coil regions next to the head domain (Fig. 1B, C). In addition, 3 we fused GFP to the C-terminus, which does not affect the function of 4 Kinesin-5, as previously reported [11]. These constructs were expressed as 5 transgenes in levels comparable to the endogenous allele with a ubiquitously 6 active promoter, as assayed by western blot (Fig. 1D). Due to the C-terminal 7 GFP moiety, the constructs showed a slower mobility in SDS-PAGE than wild 8 were lysed about 30 min after injection and extracts analyzed by western blot 10 against the C-terminus of Kinesin-5. The observed difference in 11 electrophoretic mobility was consistent with proteolytic cleavage at the TEV 12 sites at the neck and corresponding loss of the head domain. As we detected a 13 single band, proteolytic cleavage was close to complete under our 14 experimental conditions (Fig. 2E). 15 16

Cleavage of Kinesin-5 leads the loss-of-function in mitosis 17
Next we analyzed the functional consequences of the Kinesin-5 cleavage. To 18 track the nuclear cycles and behavior of chromosomes, we co-injected 19 fluorescent labelled histone-1 and TEV protease into Kinesin-5 null embryos 20 expressing the Kin-5[G394tev]-GFP transgene. Following TEV injection, we 21 observed a failure of chromosome separation and monoastral spindles (Fig. 3). 22 These phenotypes were observed in individual spindles interspersed between 23 normally appearing spindles. These phenotypes were consistent with the 24 previously reported mitotic defects following Kinesin-5 antibody injection [5]. 25

Interphase function of Kinesin-5 26
An interphase function of Kinesin-5 has not been investigated, yet. In 27 interphases of syncytial embryos, Kinesin-5-GFP is strongly enriched at the 28 centrosomes and associated asters. In addition, dynamic extended structures 1 between adjacent asters were detected (Fig. 4C). These transient signals may 2 represent microtubules coated with Kinesin-5 and possibly antiparallel aligned 3

microtubules. 4 5
As hypothesized previously [12,13], Kinesin-5 may be involved in nuclear 6 positioning and formation of the nuclear array in syncytial Drosophila embryos. 7 Kinesin-5 bound to anti-parallel aligned microtubules may push adjacent 8 asters away from each other and thus generate a repulsive force, which may 9 lead to uniform internuclear distances. In this model, Kinesin-5 would promote 10 movements of centrosome and their associated asters. Alternatively, Kinesin-5 11 may crosslink microtubules from adjacent asters and stabilize the syncytial 12 microtubule network. In this model Kinesin-5 would suppress movement of 13 centrosomes and associated asters (Fig. 4B). To distinguish these two models, Although expressed, a function of Kinesin-5 during interphase has been 4 unknown, partly because such an interphase function was obscured by the 5 mitotic defects in Kinesin-5 depleted embryos. The problem that one 6 phenotype obscures other phenotypes is common to proteins with widespread 7 functions, such as molecular motors. To circumvent this problem, we 8 developed a method for conditionally inactivating Kinesin-5. In addition to 9 Kinesin-5, this method is potentially suitable for other motor proteins, as well. 10 With a "molecular guillotine", we specifically inactivated Kinesin-5 by 11 administration of TEV protease. In this way, we revealed an interphase 12 function for the stabilization of the syncytial microtubule network. In syncytial 13 embryos, the microtubule asters originating from centrosomes can directly 14 interact with neighboring asters, since they are not physically separated by 15 plasma membranes. These interactions lead to formation of an extended 16 network covering the embryonic cortex. The phenotypic behavior of 17 centrosomes and their associated nuclei reflect their intrinsic properties but 18 also, as part of the network, the influences from the neighbors. Adjacent 19 microtubule asters potentially interact via crosslinkers such as Feo/Ase1p, 20 bundling proteins or motors with sliding activity, such as Kinesin-5. Here we 21 tested the hypothesis that Kinesin-5 generates repulsive forces between 22 adjacent astral microtubules in interphase. We expected that a loss of force 23 generation would have led to a reduced mobility of the network and its nodes, 24 the centrosomes. Using the fluctuations of centrosomes as an indicator of 25 network dynamics, we rejected our hypothesis, because we measured an 26 increased mobility of the centrosomes, when Kinesin-5 was inactivated. We 27 interpret this data in that the in vivo function of Kinesin-5 as a crosslinker is 28 more dominant than its function for sliding of anti-parallel aligned microtubules 1 and thus pushing apart adjacent microtubule asters. The in vivo function of 2 Kinesin-5 is similar to Kinesin-1, which is enriched at the cortex and F-actin 3 and actin caps. Both may be involved in anchoring microtubule asters to the 4 cortex and in this way counteract fluctuation movements of centrosomes. In summary, the novel approach of a "molecular guillotine" enabled us to 1 investigate a specific function of the motor protein Kinesin-5 in interphase. 2 Potentially, the decapitation approach can be correspondingly applied to other 3 kinesin motors as well as dyneins and myosins, as they have a related domain 4 structure in common. Images were recorded with a Zeiss microscope equipped with a spinning disc 1 (25x/NA0.7 multi immersion, 40x/NA1.3oil). Centrosome movement was 2 recorded in Sas6-GFP expressing embryos as previously described with a 3 frame rate of 1 Hz [13]. Kin-5-GFP distribution in interphase was recorded with 4 a confocal microscope (Zeiss LSM780 with airy scan unit, 63xNA1.4/oil). 5 Images were processed with Fiji/ImageJ.