The meiotic LINC complex component KASH5 is an activating adaptor for cytoplasmic dynein

Dynein-driven chromosome movement facilitates chromosome synapsis in prophase I of meiosis, which is essential for genetic exchange and completion of meiosis. Dynein-1 is recruited by KASH5 in the outer nuclear envelope. The authors show that KASH5 is an activating dynein adaptor and characterize the KASH5–dynein interaction.


Introduction 49
To conceive healthy offspring, a paternal sperm and maternal egg must be created, which 50 requires the specialised form of cell division, meiosis, in which maternal and paternal 51 homologues pair in prophase I to allow the swap of genetic material by synapsis, generating 52 genetically distinct haploid daughter cells. This is facilitated by chromosomes attaching to 53 the nuclear envelope (NE), usually via their telomeres (Kim et al., 2022;Rubin et al., 2020). 54 In vertebrates and many other organisms, the chromosomes move along the inner nuclear Since IC2 depletion reduced total cellular dynactin p150 levels by ~25% (Fig. S2 A), we used 239 a dominant negative approach as another way of testing the effect of disrupting IC2-p150 240 interactions on recruitment to KASH5, using over-expression of the coiled coil 1 region of 241 p150 (CC1) (King et al., 2003;Quintyne et al., 1999). Unfortunately, transiently transfected 242 constructs would not co-express with GFP-KASH5 in the inducible GFP-KASH5 cell line. We 243 therefore transiently co-transfected HeLa cells with GFP-KASH5 and myc-SUN2 (to enhance 244 localisation of KASH5 to the NE) along with RFP-tagged CC1. Notably, while CC1 expression 245 had no effect on dynein or LIS1 recruitment to KASH5, it prevented dynactin accumulation 246 at the NE (Fig. 2 B), confirming that dynein and LIS1 can associate with KASH5 independently 247 of dynactin. 248

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We used RNAi to investigate the involvement of LICs in the dynein-KASH5 interaction. There 250 was no reduction in the proportion of cells with detectable dynein, dynactin or LIS1 251 recruited to KASH5 after depleting LIC2 alone (Fig. 3 B, D). LIC1 depletion also had very little 252 effect on dynactin or LIS1 recruitment in a binary scoring assay, but had a variable effect on 253 dynein intermediate chain, with 80.8 ± 24.3% of cells (n=3 experiments, ±SD) showing IC 254 signal at the NE (Fig. 3 A, D). However, when both LIC1 and LIC2 were depleted 255 simultaneously, the proportion of cells with detectable dynein and dynactin at the NE was 256 reduced by ~75% (Fig. 3 C, D). LIS1 recruitment was also reduced, with ~40% of KASH5 257 expressing cells showing no LIS1 signal at the NE. We were not able to deplete endogenous 258 LIC1 completely using RNAi (Fig. S2 B) which may explain why in some cells a residual level 259 of dynein, dynactin and LIS1 remained with KASH5. In addition, GFP-KASH5ΔK pull-downs 260 from LIC1 and 2 depleted HeLa cells contained very little dynein and dynactin (Fig. 3 E   The discrepancy between the anti-LIC1 and IC scoring most likely reflects the weaker NE 271 labelling seen with the IC74 antibody compared to anti-LIC1 antibodies in control cells. Our 272 interpretation of these data is that LIS1 depletion reduces dynein levels at the NE 273 somewhat, to levels that are still detectable by anti-LIC1 but sometimes not by IC74. 274 Altogether, these data reveal that LIS1 is vital for the dynactin complex to be recruited to 275 to several dynein adaptors using purified proteins (Celestino et al., 2019). We generated the 294 helix 1 deletion and mutations in GFP-LIC1 (Fig. 4A), and also a deletion lacking helix 1 plus 295 some linker sequence (S433-S458). These were co-expressed with HA-tagged KASH5DK in 296 HeLaM cells depleted of both LICs. Full length GFP-LIC1 pulled down KASH5DK effectively, 297 whereas the LIC1 helix 1 deletions and point mutants did not (Fig. 4C). We then examined 298 the recruitment of LIC1 to KASH5 at the NE in HeLaM cells co-expressing HA-KASH5, myc-299 SUN2 and GFP-LIC1 constructs. Both wild-type GFP-LIC1 and the mid-length LIC1-CT2 were 300 recruited to KASH5, although CT2 detection at the NE was variable (Fig. 4 D, E). In contrast, 301 LIC1-CT3, the helix 1 deletions, and helix 1 point mutants were poorly recruited. Altogether, 302 these results demonstrate LIC1 helix 1 is essential for interaction with KASH5. 303

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The importance of LIC helix 1 in cargo binding prompted us to assess the role of the LICs on 305 endogenous membrane cargoes, whose adaptors have not been fully defined. First, we 306 remaining localised in the cell periphery, and the Golgi apparatus being fragmented and 332 scattered (Fig. 5 E). Moreover, GFP-LIC1-CT3 did not interact with RILP, whereas GFP-LIC1-333 CT2 or full-length GFP-LIC1 were robustly recruited to RILP positive organelles (Fig. S4 E)  phenotypes indicative of a loss of dynein function (Fig. 6 A). In contrast, expression of a 360 cytoplasmic KASH5 construct lacking its N terminal dynein binding domain ( Fig. 1 B, GFP-361 KASH5ΔNDΔK (Horn et al., 2013)) had no effect on Golgi apparatus or lysosome distribution 362 and morphology (Fig. 6 A) were expressed (Fig. 6 B). We investigated if the same was true for dynein recruitment to 373 RILP, even though RILP is not thought to be able to activate dynein/dynactin motility (Lee et  terminal region. SEC-MALS analysis showed that all three proteins formed dimers (Fig. 7 B). 387 Of these, KASH51-460 was the most active in motility assays using dynein containing a heavy 388 chain mutant that cannot form the inhibited Phi conformation (Zhang et al., 2017) (Fig. 7C). 389 We next used assays with wild type purified dynein, which when combined with dynactin 390 and LIS1 alone displayed very little motility (Fig. 7 D). The inclusion of KASH51-460 promoted 391 processive dynein movements (Fig. 7 D, E), although a lesser number than seen with 392 purified Hook31-522 (Urnavicius et al., 2018). KASH5 EF-hands (amino acids 1-115), the coiled 393 coils (amino acids 155-349), or a combination of EF-hands and coiled coils alone (amino 394 acids 1-349) were not enough to activate dynein/dynactin (Fig. 7 F). Importantly, the 395 velocity of processive dynein movements was the same for KASH51-460 and Hook31-522 ( Fig. 7  ninein, lacks some key consensus calcium-binding amino acids (at positions X,Y,Z,-X,-Y,-Z), 406 with these changes being consistent across species (Fig. 8 A). EF hand 1 is particularly 407 divergent, with only the residue in position X (Grabarek, 2006) matching the consensus, and 408 with the key position -Z being a glutamine or histidine instead of a glutamate residue. While 409 EF hand 2 has consensus amino acids in position X, other residues either do not conform or 410 vary between species. For example, in non-rodent KASH5 EF hand 2, there is aspartate in 411 place of glutamate at -Z, which can result in magnesium binding (Grabarek, 2006) S5). This recruitment was not affected by treating cells with the cell permeable calcium 420 chelator, BAPTA-AM, for 2 hours to deplete intracellular calcium (Fig. S5). Furthermore, 421 endogenous LIC1 was present at equal levels when GFP-KASH5-ND was pulled down from 422 cell lysates in the presence or absence of EGTA (Fig. 8 B). This demonstrates that the KASH5-423 dynein interaction does not require calcium and confirms that the Rab11-FIP3-dynein 424 interaction in cells is calcium-independent, as reported for in vitro assays (Lee et al., 2020). corresponding mutation in human KASH5 changes a valine to glutamic acid in EF hand 1 431 (V54E: Fig. 8 A). We generated KASH5-EF-fue constructs to establish how this mutation 432 affected KASH5-dynein interactions. We also mutated the amino acids in positions X and Y we replaced some KASH5 residues by those found in CRACR2a (Fig. 8 A). KASH5-EF-mod1 436 has four substitutions: Q46E; Q55D; P87Y and K88L. In KASH5-EF-mod2, nine amino acids in 437 EF hands 1 and 2 and part of the exiting helix were changed to the CRACR2a sequences. 438

439
To test which KASH5 mutants could form a stable complex with dynein and dynactin, GFP-440

KASH5DK-WT or EF hand mutants were isolated from HeLaM cells by GFP-trap and probed 441
for endogenous IC and dynactin p150. Unmodified GFP-KASH5ΔK co-precipitated with 442 dynein and dynactin, as did GFP-KASH5DK-EF-mod1 ( Fig. 8 B). Interestingly, dynein 443 recruitment to EF-mod1 was greater than to KASH5DK-WT in 2/3 experiments. In contrast, 444 much less dynein and dynactin bound to the GFP-KASH5DK-EF-fue, EF-AA, or EF-mod2 445 mutants. In agreement, we found that endogenous dynein was recruited to full length GFP-446 KASH5 and GFP-KASH5-EF-mod-1 at the NE, whereas GFP-KASH5-EF-fue, EF-AA, or EF-mod2 447 mutants did not accumulate dynein ( As another means of assessing the effects of these mutations on KASH5-dynein binding, we 450 harnessed the dominant negative effect of expressing cytosolic KASH5, which causes Golgi 451 fragmentation by sequestering dynein (Fig. 6A). In this assay, any mutant that prevents 452 KASH5 from binding dynein would have no effect on Golgi morphology when expressed, as 453 seen with GFP alone (Fig. 8 E). Overexpression of GFP-KASH5ΔK or GFP-KASH5ΔK-mod1 in 454 HeLaM cells resulted in strong fragmentation of the Golgi apparatus. In contrast, GFP-455 KASHΔK-EF-fue, GFP-KASHΔK-EF-AA or GFP-KASHΔK-EF-mod2 ( Fig. 8 E) had a much weaker 456 effect on Golgi positioning, implying that these EF hand mutants were much less able to 457 sequester dynein. Altogether, these findings show that the KASH5 EF hand is critical for its 458 function with dynein and dynactin, although the interaction is not calcium dependent. Our data suggest that LIS1 is essential for recruiting dynactin to KASH5 (Fig. S1). Surprisingly, 504 LIS1 depletion had much less effect on dynein recruitment. We also saw that interfering 505 with dynein IC-p150 interactions by IC2 depletion or over-expression of p150 CC1 did not 506 prevent dynein recruitment to KASH5. Based on these data, we propose that the first step in 507 KASH5 adaptor complex assembly is an interaction between LIC helix 1 and the KASH5 EF-508 hands. Next, binding of LIS1 opens the dynein phi complex. The third step involves 509 recruitment of the dynactin complex initiated by the IC-p150 interaction, followed by the 510 formation of extensive contacts between the dynactin complex and the adaptor 511 calcium depletion (Fig. S5), and the KASH5 EF-hands lack key residues known to be essential 536 for calcium binding (Fig. 8 A). Furthermore, helix 1 peptide binding to purified KASH5 was 537 not calcium sensitive, and KASH5 did not bind calcium (Agrawal et al., 2022). However, the 538 structure of the KASH5 EF-hand is clearly vital, since mutating the X and Y positions of both 539 EF-hands to alanine ablated KASH5-dynein interactions (Fig. 8). Similar adverse effects on 540 dynein-KASH5 association were seen after mutation of other residues in KASH5 EF-hands, As well as characterising KASH5 as a dynein adaptor, we also provide insight into LIC 554 function in cells. Either LIC can recruit dynein to KASH5 in cells (Fig. 3), as confirmed by using 555 pull-downs with GFP-LIC1 (Figs. 4, 8) and reconstituting KASH5-activated motility of dynein 556 containing only LIC2 (Fig. 7). Similarly, LICs 1 and 2 act redundantly for recruitment of dynein 557 to RILP, and in the positioning of the Golgi apparatus and recycling, early and late 558 endosomes in cells (Figs. 5 and S3). We also show that the helix 1-containing region is 559 essential for all these roles. This is in keeping with roles for Hooks and BicD2 at the Golgi Altogether, we have shown that KASH5 is a novel trans-membrane member of the dynein 578 activating adaptor protein class, mapped its interaction with dynein LICs, and demonstrated 579 that the KASH5 EF hands are critical for this process. This work also sheds light on order in 580 which dynein-dynactin-adaptor complexes assemble in cells, and the involvement of LIS1 in 581 this process. 582

Materials and Methods 583
Antibodies and constructs 584 The following mouse antibodies were used: dynein IC (IC74, MAB1618, Millipore; 2mM MgCl2, pH 6.9) containing 0.5% Triton X-100. Cells were then transferred to PHEM 777 buffer supplemented with 3.7% paraformaldehyde for 20 minutes. This was followed by 778 incubation in 0.2% Triton X-100 in PBS for 5 minutes. Coverslips were rinsed in PBS and 779 quenched with glycine before labelling.  knowing the sample identity. Cells with strong nuclear enrichment of GFP-KASH5 and HA-805 SUN1 (Fig. 8) or GFP-KASH5 and myc-SUN2 (Fig. 4), but with limited accumulation in the ER, 806 were selected without reference to the LIC1 channel, which was then used to score for 807 dynein recruitment. For these data, statistical tests were not deemed appropriate as there 808 Optilab® T-rEX™ differential refractometer (Wyatt Technology). Differential refractive index 848 and light scattering data were collected and analysed using ASTRA® 6 software (Wyatt 849 Technology). Molecular weights and estimated errors were calculated across eluted peaks 850 by extrapolation from Zimm plots using a dn/dc value of 0.1850 ml g −1 . Bovine serum 851 albumin (ThermoFisher) was used as the calibration standard.  Table S1. plotted. Statistical analysis was performed using multinomial logistic regression (see Table  1078 S2  Statistical analysis is given in Table S3.