Kinesin-1 autoinhibition facilitates the initiation of dynein cargo transport

Kinesin-1s are plus-end-directed microtubule motors that transport various cargoes, and kinesin-1 defects cause many human diseases (Boecker et al., 2021; Brenner et al., 2018; Gennerich and Vale, 2009; Hannaford et al., 2022; Hirokawa and Tanaka, 2015; Kelliher et al., 2018; Keren-Kaplan and Bonifacino, 2021; Kruppa and Buss, 2021; Nicolas et al., 2018; Roney et al., 2021). The activity of kinesin-1 is regulated by multiple factors, including its light chains, cargo adapter proteins, an opposite motor that binds to the same cargo, phosphorylation, and microtubule-binding proteins (Ally et al., 2009; Balabanian et al., 2022; Blasius et al., 2007; Byrd et al., 2001; Chen and Sheng, 2013; Fenton et al., 2021; Ferro et al., 2022; Fu and Holzbaur, 2013; Gindhart et al., 1998; Guardia et al., 2021; Keren-Kaplan and Bonifacino, 2021; Monroy et al., 2020; Sakamoto et al., 2005; Twelvetrees et al., 2019; Verhey and Hammond, 2009; Williams et al., 2014; Xu et al., 2012; Zhao et al., 2021). An important aspect of kinesin-1 regulation is its autoinhibition that involves an interaction between the C-terminal tails and motor domains (Cai et al., 2007; Chiba et al., 2022; Coy et al., 1999; Dietrich et al., 2008; Friedman and Vale, 1999; Kaan et al., 2011; Seiler et al., 2000; Stock et al., 1999; Weijman et al., 2022; Wong et al., 2009). Recently, a KIF5A (kinesin-1) mutation causing the neurodegenerative disease Amyotrophic Lateral Sclerosis (ALS) has been linked to a loss of autoinhibition (Baron et al., 2022; Nakano et al., 2022; Pant et al., 2022). However, the importance of kinesin-1 autoinhibition in normal cells needs to be dissected.

Cytoplasmic dynein is the minus-end-directed microtubule motor transporting many cargoes (Reck-Peterson et al., 2018), and defects in dynein and/or its regulator dynactin are implicated in ALS or ALS-like motor neuron degeneration (Chevalier-Larsen and Holzbaur, 2006; Gershoni-Emek et al., 2016; Hafezparast et al., 2003; Ikenaka et al., 2013; Kieran et al., 2005; Lai et al., 2007; LaMonte et al., 2002; Maimon et al., 2021; Mentis et al., 2022; Münch et al., 2004; Stavoe and Holzbaur, 2019; Ström et al., 2008; Yu et al., 2018). Some vesicular cargoes bind dynein and kinesin-1 through the same cargo adapter (Celestino et al., 2022; Cox and Spradling, 2006; Fenton et al., 2021; Fu and Holzbaur, 2014; Canty et al., 2021,Preprint), and in this case, loss of kinesin-1 autoinhibition may overcome dynein to change cargo distribution (Baron et al., 2022; Kelliher et al., 2018). In many cell types, dynein itself is a cargo of kinesin-1 (Arimoto et al., 2011; Brendza et al., 2002; Duncan and Warrior, 2002; Egan et al., 2012; Hirokawa et al., 1990; Januschke et al., 2002; Lenz et al., 2006; Twelvetrees et al., 2016; Yamada et al., 2010; Zhang et al., 2003). In mammalian neurons, kinesin-1 interacts with dynein directly through kinesin light chains and moves dynein towards microtubule plus ends (Ligon et al., 2004; Twelvetrees et al., 2016). In filamentous fungi, kinesin-1s are important (although not essential) for the microtubule plus-end accumulation of dynein and dynactin (Egan et al., 2012; Lenz et al., 2006; Peñalva et al., 2017; Yao et al., 2012; Zhang et al., 2003; Zhang et al., 2010). Fungal kinesin-1s do not have associated light chains (Seiler et al., 2000; Steinberg and Schliwa, 1995), but dynactin is important for the dynein-kinesin-1 interaction (Qiu et al., 2018). The kinesin-1-mediated dynein accumulation at the plus ends is important for the retrograde transport of dynein cargoes such as early endosomes (Abenza et al., 2009; Lenz et al., 2006; Zekert and Fischer, 2009). Plus-end-directed transport of early endosomes does not use kinesin-1 but uses kinesin-3 (Lenz et al., 2006; Wedlich-Söldner et al., 2002; Zekert and Fischer, 2009). Dynein transports early endosomes away from the plus ends with the help of dynactin and the FTS-Hook-FHIP complex (Bielska et al., 2014; Yao et al., 2014; Zhang et al., 2011b; Zhang et al., 2014). The mechanism of dynein-mediated early endosome transport is largely conserved and has been dissected in detail using mammalian proteins (Christensen et al., 2021; Guo et al., 2016; Lau et al., 2021; Olenick et al., 2019; Schroeder and Vale, 2016; Urnavicius et al., 2018; Yeh et al., 2012). However, factors regulating this process require further studies. Here, we present our work identifying kinesin-1 autoinhibition as a new factor important for the initiation of dynein-mediated early endosome transport.

In Aspergillus nidulans, microtubule plus ends face the hyphal tip, and minus ends are at either the spindle-pole body or septum (Efimov et al., 2006; Egan et al., 2012; Han et al., 2001; Oakley et al., 1990; Xiong and Oakley, 2009; Zeng et al., 2014; Zhang et al., 2017b). The plus-end accumulation of dynein is represented by the comet-like structures of GFP-dynein near the hyphal tip (Han et al., 2001; Xiang et al., 2000). A defect in dynein-mediated transport causes an abnormal hyphal-tip accumulation of early endosomes and their hitchhiking cargoes (Abenza et al., 2009; Lenz et al., 2006; Salogiannis and Reck-Peterson, 2017; Zekert and Fischer, 2009). Genetic screens for early-endosome distribution (eed) mutants allowed the identification of novel dynein regulators (Xiang and Qiu, 2020). Here, we performed a UV mutagenesis using a strain containing mCherry-RabA-marked early endosomes and GFP-labeled dynein heavy chain (Pinar and Peñalva, 2021; Zhuang et al., 2007). In the eedE16 mutant isolated after mutagenesis, we observed plus-end GFP-dynein comets at the hyphal tip (Fig. 1 A). Diffuse signals near the comets possibly contributed to an increase in the hyphal-tip GFP-dynein signal intensity (Fig. 1, A and B). Early endosomes (mCherry-RabA) abnormally accumulated near the hyphal tip (Fig. 1, A and C).

The eedE16 mutation was identified through whole-genome sequencing, and it is in kinA, which encodes the only kinesin-1 in A. nidulans (Requena et al., 2001). The mutation substituted an A with T that changed the codon for lysine (K; AAG) to a stop codon (TAG) at residue 895 of KinA, a 927-aa protein. This results in the deletion of 33 amino acids at the C-terminus of KinA. The C-termini of Drosophila and human Kinesin-1s contain two important regions: the IAK motif involved in autoinhibition (Kaan et al., 2011), and the RKRYQ region responsible for ATP-independent microtubule binding (Lu et al., 2016; Winding et al., 2016). The IAK motif but not the RKRYQ region is conserved in KinA (Fig. S1). K895 corresponds to K within the IAK motif (Fig. 1 D and Fig. S1). Since this motif is key to autoinhibition while additional C-terminal residues may further stabilize the autoinhibited structure (Hackney and Stock, 2000; Kaan et al., 2011), the kinA^K895* mutation may eliminate kinesin-1 autoinhibition.

The phenotype of the kinA^K895* mutant differs from that of the ∆kinA mutant (Requena et al., 2001). Loss of kinA causes dynein to localize along microtubules and decreases its plus-end accumulation near hyphal tip (Egan et al., 2012; Peñalva et al., 2017; Zhang et al., 2010), but kinA^K895* causes a stronger hyphal-tip dynein accumulation (Fig. 1, A and B). The kinA^K895* mutant colony is smaller than a wild-type colony but significantly bigger than the ∆kinA colony (Fig. 1, E and F). Thus, the normal kinesin-1 function is partially retained in the kinA^K895* mutant.

Since microtubule polarity looked normal in the kinA^K895* mutant (Fig. S2 A; and Videos 1 and 2), the hyphal-tip accumulation of early endosomes indicates a dynein defect. Our quantitation showed a significant decrease in the frequency but not the speed of dynein-mediated transport in the mutant (Fig. 1, G–I). The frequency but not the speed of plus-end-directed transport is also lowered (Fig. 1, J and K), although the movement is driven by kinesin-3 rather than kinesin-1 (Lenz et al., 2006; Wedlich-Söldner et al., 2002; Zekert and Fischer, 2009). This has been similarly observed in other dynein-pathway mutants (Egan et al., 2012; Zhang et al., 2011b, 2018), which is likely caused by kinesin-3 having a lower chance to meet the cargoes abnormally blocked at the hyphal tip. The kinA^K895* mutation also causes a mild but noticeable nuclear-distribution defect (Fig. S2, B, C, and D), consistent with an effect on the function of dynein in nuclear movement (Xiang, 2018).

To compare the wild-type and kinA^K895* kinesin-1s, we replaced the wild-type kinA gene with either the kinA-GFP or kinA^(1–894)-GFP fusion gene. The kinA^(1–894)-GFP mutant formed a colony similar to that of the kinA^K895* mutant (Fig. 2, A and B). KinA-GFP formed a diffuse background in the cytoplasm with occasional faint hyphal-tip signals, consistent with its autoinhibition, but KinA^(1–894)-GFP formed a strong hyphal-tip accumulation at and/or near microtubule plus ends (Fig. 2, C and D), consistent with the localization of uninhibited kinesin-1s in other cell types (Baron et al., 2022; Seiler et al., 2000; Twelvetrees et al., 2016; Weijman et al., 2022). This accumulation was not affected by the lack of dynein heavy chain (alcA-nudA^HC; Fig. 2, E and F). In the kinA^(1–894)-GFP mutant, early endosomes (mCherry-RabA) accumulated abnormally near the hyphal tip (Fig. 2, C and G), just like in the kinA^K895* mutant (Fig. 1, A and C).

To determine whether the defect in dynein-mediated early endosome transport is caused by a loss of IAK-mediated autoinhibition or by the loss of other deleted C-terminal amino acids that may regulate dynein, we made the kinA^∆IAK-GFP and the kinA^K895E-GFP mutants. These mutants looked similar to the kinA^(1–894)-GFP mutant on plates (although their colonies are slightly bigger; Fig. 3, A and B), and they also exhibited a strong hyphal-tip accumulation of KinA-GFP (Fig. 3, C and D). Importantly, early endosomes accumulated abnormally near the hyphal tip in these mutants (Fig. 3, C and E), indicating that the IAK motif plays a key role in dynein-mediated early endosome transport.

Several amino acids in the motor domain of Drosophila KHC have been implicated in autoinhibition (Kaan et al., 2011; Kelliher et al., 2018). For example, D185 may interact with the IAK motif (Kaan et al., 2011), and E177K relieves autoinhibition in neurons (Kelliher et al., 2018). Since E186 of KinA corresponds to D185 of Drosophila KHC (Fig. S1), we constructed the kinA^E186K-GFP strain. We also tried to construct a kinA^E178K-GFP mutant (corresponding to E177K in Drosophila KHC; Fig. S1) as well as a kinA^E178K,E186K-GFP mutant. However, the kinA^E178K-GFP mutant we intended to make contained an extra mutation (P426L), and the kinA^E178K,E186K-GFP mutant also contained an extra mutation (K735R). As the extra mutation in either case is in a non-conserved amino acid (Fig. S1), and K735R does not change the charge, we studied all these mutants further. Interestingly, E178K had almost no effect (Fig. 3, A, B, and F–H), possibly due to KinA being subtly different from Drosophila KHC in structure. While E186K only had a moderate effect (Fig. 3, A, B, and F–H), the kinA^{E178K,E186K,K735R}-GFP mutant almost phenocopied the kinA^(1–894)-GFP mutant: It formed a colony similar to that of the kinA^(1–894)-GFP mutant (Fig. 3, A and B), and it also exhibited a strong hyphal-tip KinA-GFP accumulation (Fig. 3, F and G) and an abnormal early endosome accumulation (Fig. 3, F and H). As kinesin-1s are normally autoinhibited and their strong accumulation at cell periphery near microtubule plus ends has been linked to a loss of autoinhibition in different cell types (Baron et al., 2022; Kelliher et al., 2018; Seiler et al., 2000; Twelvetrees et al., 2016; Weijman et al., 2022), our results suggest strongly that the defect in dynein-mediated early endosome transport is caused by a loss of kinesin-1 autoinhibition.

In the kinA^K895* mutant, the overall intensity of GFP-dynein is higher than normal near the hyphal tip (Fig. 1 B), suggesting that dynein can be transported there but cannot leave. In the ∆hookA mutant lacking the early endosomal dynein adapter, dynein does not leave the plus end with its cargo, and its hyphal-tip intensity is higher than in wild type regardless of whether the kinA^K895* mutation is present (Fig. S2 E). In the kinA^K895*, ∆hookA double mutant, the plus-end dynein comet intensity is similar to that in the ∆hookA single mutant (Fig. 4, A and B), suggesting that kinesin-1 autoinhibition is not essential for the plus-end dynein accumulation. This is consistent with a previous notion that the posterior localization of dynein during Drosophila oogenesis needs kinesin-1 but not its IAK region for autoinhibition (Williams et al., 2014).

In filamentous fungi, kinesin-1 and myosin-V are both able to transport secretory vesicles to support hyphal tip extension (Pantazopoulou et al., 2014; Peñalva et al., 2017; Pinar et al., 2022; Schuchardt et al., 2005; Schuster et al., 2012). In A. nidulans, the ∆kinA, ∆myoV, or alcA-myoV (myosin-V conditional-null) single mutant forms a colony smaller than a wild-type colony (Requena et al., 2001; Taheri-Talesh et al., 2012; Zhang et al., 2011a). However, the ∆kinA, alcA-myoV double mutant is nearly inviable when grown on glucose that represses myoV expression (Zhang et al., 2011a; Fig. 4 C), and the ∆kinA, ∆myoV double mutant is also nearly inviable (Peñalva et al., 2017). In contrast, colony of the kinA^(1–894)-GFP, alcA-myoV double mutant is even bigger than that of the alcA-myoV single mutant, and colony of the kinA^(1–894)-GFP, ∆myoV double mutant is similar to that of the ∆myoV single mutant (Fig. 4 D). Despite the subtle difference in the genetic interactions involving the null allele verses the conditional-null allele of myosin-V, our data strongly indicate that kinesin-1 autoinhibition is not essential for the transporting function of kinesin-1 in supporting hyphal growth. Consistent with this notion, RabE (Rab11)-marked secretory vesicles, which are cargoes of kinesin-1 and myosin-V (Pantazopoulou et al., 2014; Peñalva et al., 2017; Pinar et al., 2022), reach the hyphal tip in the kinA^K895*, ∆myoV double mutant (Fig. 4, E and F), and the hyphal-tip intensity of RabE-GFP is even higher in the double mutant than that in the ∆myoV mutant (Fig. 4 F).

RabE-marked secretory vesicles are cargoes of kinesin-1 and dynein (Pantazopoulou et al., 2014; Peñalva et al., 2017), although the cargo adapter has yet to be identified. These vesicles are normally tethered at the hyphal tip by the actin cytoskeleton, but in the ∆myoV background or upon loss of the actin cytoskeleton, their bidirectional movements can be observed (Pantazopoulou et al., 2014; Peñalva et al., 2017). In the kinA^K895*, ∆myoV double mutant, both the frequency and the speed of dynein-mediated movement are significantly reduced compared to the ∆myoV single mutant (Fig. 4, G, H, and I). This differs from the result on early endosomes because only the frequency but not the speed of dynein-mediated early endosome movement is reduced in the kinA^K895* mutant (Fig. 1, H and I). Possibly, as kinesin-1 drives the plus-end-directed movement of the RabE vesicles, its constitutive activation directly competes against dynein-mediated movement of the same cargo, and similar phenomena might occur in other systems (Baron et al., 2022; Kelliher et al., 2018). The frequency but not the speed of plus-end-directed RabE-vesicle movement is also lower in the presence of kinA^K895* (Fig. 4, J and K), just like in the case of early endosomes whose plus-end-directed transport is driven by kinesin-3 rather than kinesin-1 (Fig. 1, J and K; Egan et al., 2012; Lenz et al., 2006; Wedlich-Söldner et al., 2002; Zekert and Fischer, 2009), most likely due to the lack of cargoes as they are being abnormally held at the hyphal tip.

In the kinA^K895* mutant, we observed a decrease in the frequency but not the speed of dynein-mediated early endosome movement, which is conceptually similar to the defect observed in a mutant lacking NudF/LIS1 (although the defect in the LIS1 mutant is much more severe; Egan et al., 2012). LIS1 is a conserved dynein regulator that binds to the dynein motor domain and plays a specific role in dynein activation (Gillies et al., 2022; Markus et al., 2020; Reimer et al., 2022,Preprint). Dynein activation involves conformational changes of the dynein heavy chain dimer from an auto-inhibited “phi” conformation to the open conformation (Torisawa et al., 2014; Zhang et al., 2017a), and binding of dynactin and cargo adapter to the dynein tails turns dynein to a parallel conformation needed for processive motility (Zhang et al., 2017a). The phi mutation disrupts the phi structure and keeps dynein open, thereby facilitating dynein activation (Zhang et al., 2017a). In A. nidulans, the phi mutation (nudA^R1602,K1645E) significantly reduces the abnormal hyphal-tip accumulation of early endosomes in the temperature-sensitive nudF6 (lis1) mutant (Fig. 5, A–C; Qiu et al., 2019), suggesting a role of LIS1 in overcoming the autoinhibited conformation (Qiu et al., 2019), similarly observed in other systems especially budding yeast (Elshenawy et al., 2020; Gillies et al., 2022; Htet et al., 2020; Marzo et al., 2020; Karasmanis et al., 2022 Preprint). However, the phi mutation does not significantly affect the hyphal-tip accumulation of early endosomes in the kinA^K895* mutant (Fig. 5, A–C). Thus, kinesin-1 autoinhibition must be involved in a step of dynein-mediated transport beyond switching dynein to the open state.

Cargo adapters activate dynein to leave the microtubule plus ends both in vitro and in cultured cells (Baumbach et al., 2017; Jha et al., 2017; Lammers and Markus, 2015; McKenney et al., 2014; Qiu et al., 2019; Schlager et al., 2014; Splinter et al., 2012). In A. nidulans, overexpression of the cytosolic ∆C-HookA causes dynein to undergo a LIS1-dependent relocation from the microtubule plus ends to the minus ends on septa or spindle-pole bodies (Qiu et al., 2019). Since the spindle-pole body accumulation of activated dynein is cell-cycle dependent (Bieger et al., 2021), here we only focused on the septal minus ends (Zhang et al., 2017b). In the kinA^K895* mutant, overexpression of ∆C-HookA causes the septal accumulation of dynein, but the plus-end-to-minus-end relocation is defective as evidenced by the more easily observable plus-end dynein comets and the reduced septal signals (Fig. 5, D–F). Thus, although kinesin-1 autoinhibition is not essential for cargo-adapter-mediated dynein activation, it facilitates this process.

Kinesin-mediated transport of dynein to the microtubule plus ends has been reported from fungi to mammalian neurons (although kinesin-7 instead of kinesin-1 is used in budding yeast; Carvalho et al., 2004; Roberts et al., 2014; Twelvetrees et al., 2016; Yamada et al., 2010; Zhang et al., 2003). In several systems, the plus-end accumulation of dynein and its regulator dynactin plays an important role in cargo binding or the initiation of cargo transport (Lenz et al., 2006; Lloyd et al., 2012; Markus and Lee, 2011; Moughamian and Holzbaur, 2012; Splinter et al., 2012; Vaughan et al., 2002; Xiang and Qiu, 2020). We suggest that kinesin-1 autoinhibition facilitates dynein-mediated transport in these systems. The initiation of dynein-mediated transport needs LIS1 and its binding protein NudE, as well as the dynactin complex and cargo adapters (Egan et al., 2012; Garrott et al., 2022; Lenz et al., 2006; Markus et al., 2020; Olenick and Holzbaur, 2019; Qiu et al., 2019; Reck-Peterson et al., 2018). Kinesin-1 autoinhibition facilitates this process even after dynein opening, but how it affects dynein activation by dynactin and cargo adapters still remains a mystery. Recent studies suggest that some activated dynein complexes contain not only two dynein dimers (Grotjahn et al., 2018; Urnavicius et al., 2018) but also two cargo adapters (Chaaban and Carter, 2022). One possibility is that uninhibited kinesin-1s fail to dissociate from dynein-dynactin after delivering them to the microtubule plus ends, which could interfere with an optimal dynein-dynactin-cargo adapter interaction needed for the initiation of cargo transport. Alternatively, the uninhibited kinesin-1s may bring dynein back to the microtubule plus ends near the hyphal tip shortly after initiation of a retrograde transport event (Gicking et al., 2022). These ideas would need to be further tested.

Recent studies indicate that an ALS-causing mutation of KIF5A is a gain-of-function mutation disrupting autoinhibition (Baron et al., 2022; Nakano et al., 2022; Pant et al., 2022). In A. nidulans, kinA^K895* or kinA^(1–894)-GFP is also a gain-of-function mutation affecting dynein-mediated early endosome transport in the presence of wild-type kinA (Fig. S2, F and G). As dynein defects are linked to ALS (Chevalier-Larsen and Holzbaur, 2006; Liu and Henty-Ridilla, 2022), a defect in dynein-mediated transport could possibly contributes to the KIF5A-mutation-caused ALS. It is worthwhile to test this idea and other potential factors including aggregation of uninhibited kinesins or possible changes of microtubules (Baron et al., 2022; Budaitis et al., 2022; Chiba et al., 2022; Kawano et al., 2022; Nakano et al., 2022; Pant et al., 2022).

A. nidulans strains used in this study are listed in Table S1. UV mutagenesis on spores of A. nidulans strains was done as previously described (Willins et al., 1995; Xiang et al., 1999). Specifically, we collected some asexual spores from colony of the XX222 strain with a pipet tip containing ∼100 μl of sterilized distilled water and added the spores to 30 ml of sterilized distilled water contained in a 50-ml tube. We then poured about 25 ml of spore suspension into a Petri dish and placed the dish ∼6 in under a Spectronics UV Lamp (ENF-240C) to be treated with the short wave ultraviolet (254 nm) for 5–10 min. Different volumes (for example, 1, 10, 50 or 100 μl) of both the untreated spore suspension and the treated spore suspension were spread on plates with solid rich medium and incubated at 37°C for 2 d. A typical mutagenesis should kill about 95% of the spores, which can be determined by counting the number of colonies. From the plates with colonies from the mutagenized spores, we selected colonies (normally about 1% of the total colonies) that looked smaller and less bright in color, which is indicative of a decrease in asexual spore production. Spores from these mutant colonies were cultured for further microscopic examination on early-endosome distribution and dynein localization. The kinA^K895* mutant was obtained after we examined more than one hundred mutant colonies.

Solid rich medium was made of either YAG (0.5% yeast extract and 2% glucose with 2% agar) or YAG+UU (YAG plus 0.12% uridine and 0.11% uracil). Genetic crosses and diploid construction were done by standard methods. Solid minimal medium containing 1% glucose was used for selecting progeny from a cross and for selecting diploids. For live-cell imaging, cells were cultured in liquid minimal medium containing 1% glycerol for overnight at 32°C. All the biochemical analyses (for confirming strain genotypes) and genomic DNA preparation were done using cells grown at 32°C for overnight in liquid YG rich medium (0.5% yeast extract and 2% glucose). For experiments using strains containing the alcA-nudA^HC allele, we harvested spores from the solid minimal medium containing 1% glycerol and cultured them for imaging analysis in liquid minimal medium containing 1% glucose, which is a repressive medium for the alcA promoter. To identify the eedE16 mutation, we used whole-genome sequencing and bioinformatic service of Genewiz (www.genewiz.com), and we also used the software Integrative Genomics Viewer (IGV 2.4.3) to visualize the genomic sequencing data (Qiu et al., 2020).

Strains were constructed using standard procedures used in A. nidulans (Nayak et al., 2006; Szewczyk et al., 2006; Yang et al., 2004). For constructing the kinA-GFP fusion, we used the following six oligos to amplify genomic DNA from RQ177 (Qiu et al., 2018) that contains the GFP-AfpyrG fusion (Yang et al., 2004): K5U (5′-AGT​CTT​TCA​GAG​ACG​CAG​GG-3′), BW2 (5′-TCG​TCT​ATC​AAA​AAA​CCA​ACT​TGT​G-3′), KF3 (5′-CAC​AAG​TTG​GTT​TTT​TGA​TAG​ACG​AGG​AGC​TGG​TGC​AGG​CG-3′), BW4 (5′-CCA​TCT​AGA​TAT​CTG​CAG​GAA​GGG​GCT​GTC​TGA​GAG​GAG​GCA​CTG-3′), BW5 (5′-CCC​CTT​CCT​GCA​GAT​ATC​TAG​ATG​G-3′) and BW6 (5′-GCT​GAA​GTT​GGT​TGA​TTT​GCG​G-3′). Specifically, K5U and BW2 were used to amplify the ∼1-kb fragment in the coding region, and BW5 and BW6 were used to amplify the ∼1-kb fragment in the 3′ untranslated region, and KF3 and BW4 were used to amplify the ∼2.7-kb GFP-AfpyrG fragment using genomic DNA from the RQ177 strain (Qiu et al., 2018). We then used two oligos, K5U and BW6, for a fusion PCR of the three fragments to generate the ∼4.6-kb kinA-GFP-AfpyrG fragment that we used to transform into the XY42 strain (Qiu et al., 2018) containing ∆nkuA (Nayak et al., 2006) and mCherry-RabA (Abenza et al., 2009; Zhang et al., 2010).

For transformation, spores from XY42 were cultured in a flask containing 50 ml YG+UU liquid medium, which was shaken overnight at 80 rpm at room temperature and then at 180 rpm at 32°C for about 1.5 h. The medium was poured off, and hyphae were then treated with about 20 ml solution containing cell-wall-lysing enzymes. This solution contains 10 ml of solution 1 (52.8 g of ammonium sulfate and 9.6 g of citric acid in 500 ml water, pH adjusted to 6.0 with 5 M KOH), 10 ml of solution 2 (5 g of yeast extract and 10 g of sucrose in 500 ml water), 0.25 ml of 1 M MgSO₄, 200 mg of fraction V bovine serum albumin, 60 mg of lysing Enzymes (L1412; Sigma-Aldrich), and 0.05 ml of β-glucuronidase (G8885; Sigma-Aldrich). This mixture was made and filter-sterilized within 1 h before being used to treat the hyphae. After about 3 h of treatment with this solution at 32°C with shaking at 180 rpm, protoplasts were generated. The protoplasts were collected by centrifugation at 1,700 rpm for 1 min using a swing-bucket rotor (Eppendorf S-4-72), washed with 15 ml of ice-cold solution 3 (26.4 g of ammonium sulfate, 5 g of sucrose and 4.8 g of citric acid in 500 ml water, pH adjusted to 6.0 with 5 M KOH), and finally suspended in 0.5 ml of ice-cold solution 5 (4.48 g of KCl, 0.75 g of CaCl₂ and 0.195 g of MES in 100 ml water, pH adjusted to 6.0 with 5 M KOH). In a 15-ml tube, 100 μl protoplast was mixed with 20 μl DNA (1–2 μg total) and 50 μl ice-cold solution 4 (25 g of PEG 6000 or 8000 (P2139; Sigma-Aldrich), 1.47 g of CaCl₂.2H₂O, 4.48 g of KCl, and 1.0 ml of 1 M Tris-HCl pH 7.5 in 100 ml water). This mixture was kept on ice for 20 min, followed by addition of 1 ml solution 4 with gentle mixing. The tube was kept at room temperature for 20 min. 10 ml of 50°C pre-melted solid medium (YAG + 0.6 M KCl) was added into the tube and the mixture was poured into a petri dish with a thin layer of the same solid medium (YAG + 0.6 M KCl). After the plates were incubated at 37°C for 2–3 d, colonies of transformants appeared. Autoclaved toothpicks were used to touch the top of the individual colony and transfer the asexual spores onto a YAG plate, which was incubated at 37°C for 2 d. The transformants were then screened by microscopically observing the GFP signals, and the presence of the kinA-GFP fusion was confirmed by western blotting analysis with a polyclonal anti-GFP antibody from Clontech. In addition, we also performed a diagnostic PCR to verify the correct integration using oligos K5UTR (5′-GAA​CGA​CCT​CAC​AGA​CTC​A-3′) and AFpyrG3 (5′-GTT​GCC​AGG​TGA​GGG​TAT​TT-3′).

For constructing the kinA^(1–894)-GFP strain, we made the kinA^(1–894)-GFP-AfpyrG fragment by inserting the GFP-AfpyrG fragment into the kinA^K895* mutation site right before the stop codon. The following four oligos were used to make the kinA^(1–894)-GFP-AfpyrG construct: K5U, K895.R (5′-GCA​CCA​GCT​CCA​GCG​ATT​CGG​GAG​CCA​G-3′), K895.F (5′-AAT​CGC​TGG​AGC​TGG​TGC​AGG​CG-3′), and BW6. Specifically, K5U and K895.R were used to amplify a ∼0.9-kb fragment of the 5′ coding region, and K895.F and BW6 were used to amplify a ∼3.6-kb fragment using genomic DNA from the RQ197 strain (containing kinA-GFP) as template. We then used two oligos, K5U and BW6, for a fusion PCR to fuse the two fragments to generate the ∼4.5-kb kinA^(1–894)-GFP-AfpyrG fragment that we used to transform the XY42 strain and the RQ54 strain. The transformants were screened by microscopically observing the GFP signals and further confirmed by a Western blotting analysis with a polyclonal anti-GFP antibody from Clontech. In addition, we also performed a diagnostic PCR to verify the correct integration using oligos K5UTR (5′-GAA​CGA​CCT​CAC​AGA​CTC​A-3′) and GFP-5R (5′-CAG​TGA​AAA​GTT​CTT​CTC​CTT​TAC​T-3′).

We made the kinA^∆IAK-GFP-AfpyrG fragment using the following four oligos: K5U2 (5′-GCC​AGT​CTT​TCA​GAG​ACG​CAG​G-3′), IAK-R (5′-ACG​GAG​AGG​TCG​GGA​GCC​AGC​GAA​GCT-3′), IAK-F (5′-GGC​TCC​CGA​CCT​CTC​CGT​GGC​GGC​G-3′), and Kin3R (5′-GCT​GAA​GTT​GGT​TGA​TTT​GCG​GAC-3′). Specifically, K5U2 and IAK-R were used to amplify a ∼0.9-kb fragment of the 5′ coding region, and IAK-F and Kin3R were used to amplify the ∼3.7-kb fragment using genomic DNA from the RQ197 strain (containing kinA-GFP) as template. We then used two oligos, K5U2 and Kin3R, for a fusion PCR to fuse the two fragments to generate the ∼4.6-kb kinA^∆IAK-GFP-AfpyrG fragment that we used to transform the XY42 strain. The transformants were screened by microscopically observing the GFP signals. In addition, we also performed a diagnostic PCR to verify the correct integration using oligos K5U3 (5′-GTT​TTC​TGA​CAA​CGA​GCG​GAA​GC-3′) and GFP5R2 (5′-GCA​TCA​CCT​TCA​CCC​TCT​CCA​C-3′), which amplified a product of ∼1.3-kb. This 1.3-kb fragment was sequenced to verify the deletion of the IAK-coding sequence in the absence of other mutations. The sequence primers used were K5U3 and GFP5R2.

We made the kinA^K895E-GFP-AfpyrG fragment using the following four oligos: K5U, K895ER (5′-CCG​CCA​CGG​AGA​GGC​TCA​GCG​ATT​CGG​GAG​CCA​GC-3′), K895EF (5′-GAG​CCT​CTC​CGT​GGC​GG-3′), and BW6. Specifically, K5U and K895ER were used to amplify a ∼0.9-kb fragment of the 5′ coding region, and K895EF and BW6 were used to amplify the 3.7-kb fragment using genomic DNA from the RQ197 strain (containing kinA-GFP) as template. We then used two oligos, K5U and Kin3R, for a fusion PCR to fuse the two fragments to generate the ∼4.6-kb kinA^K895E-GFP-AfpyrG fragment that we used to transform the XY42 strain. The transformants were screened by microscopically observing the GFP signals. In addition, we also performed a diagnostic PCR to verify the correct integration using oligos K5U3 and GFP5R2, which amplified a product of ∼1.3-kb. This 1.3-kb fragment was sequenced to verify the K895E mutation in the absence of other mutations. The sequence primers used were K5U3 and GFP5R2.

We made the kinA^E186K-GFP-AfpyrG fragment using the following four oligos: K5UTR2 (5′-TGA​ACG​ACC​TCA​CAG​ACT​CAC​TCC-3′), E186K-R (5′-TGA​CTT​CGT​AAA​CCT​TTT​GTA​CGC​TAG​AGA​C-3′), E186K-F (5′-GTC​TCT​AGC​GTA​CAA​AAG​GTT​TAC​GAA​GTC​A-3′), and KinR1 (5′-GCT​TCC​GCT​CGT​TGT​CAG​AAA​AC-3′). Specifically, K5UTR2 and E186K-R were used to amplify a ∼0.9-kb fragment, and E186K-F and KinR1 were used to amplify a ∼1.2-kb fragment using genomic DNA from the RQ197 strain (containing kinA-GFP) as template. We then used two oligos, K5UTR2 and KinR1, for a fusion PCR to fuse the two fragments to generate a ∼2.1-kb fragment. We co-transformed the ∼2.1-kb fragment with the ∼4.6-kb kinA-GFP-AfpyrG-containing fragment (amplified using K5U2 and Kin3R as primers) into the XY42 strain. The transformants were screened by microscopically observing the GFP signals. In addition, we also performed a PCR using K5UTR3 (5′-CAG​CTC​CGT​ATC​TCT​TGT​CGG​TCT-3′) and GFP5R2 as primers, and sequenced the PCR product with primers K5UTR2, KinR1, K5U3, and GFP5R2 to verify the E186K mutation in the absence of other mutations.

We made the kinA^E178K-GFP-AfpyrG fragment using the following four oligos: K5UTR2, E178K-R (5′-CTA​GAG​ACG​TAA​ACC​TTA​AGC​AGA​CCT​TTG​A-3′), E178K-F (5′-TCA​AAG​GTC​TGC​TTA​AGG​TTT​ACG​TCT​CTA​G-3′), and KinR1. Specifically, K5UTR2 and E178K-R were used to amplify a ∼0.9-kb fragment, and E178K-F and KinR1 were used to amplify a ∼1.2-kb fragment using genomic DNA from the RQ197 strain (containing kinA-GFP) as template. We then used two oligos, K5UTR2 and KinR1, for a fusion PCR to fuse the two fragments to generate a ∼2.1-kb fragment. We co-transformed the ∼2.1-kb fragment with the ∼4.6-kb kinA-GFP-AfpyrG-containing fragment (amplified using K5U2 and Kin3R as primers) into the XY42 strain. The transformants were screened by microscopically observing the GFP signals. In addition, we also performed a PCR using K5UTR3 and GFP5R2 as primers, and sequenced the PCR product with primers K5UTR2, KinR1, K5U3, and GFP5R2 to verify the E178K mutation in the absence of other mutations. However, the sequencing analysis detected the unintended P426L mutation.

We made the kinA^E178K,E186K-GFP-AfpyrG fragment using the following four oligos: K5UTR2, E178K-R, E178K-F, and KinR1. Specifically, K5UTR2 and E178K-R were used to amplify a ∼0.9-kb fragment using genomic DNA from the RQ400 strain (containing kinA^{E178K, P426L}-GFP) as template, and E178K-F and KinR1 were used to amplify a ∼1.2-kb fragment using genomic DNA from the RQ395 strain (containing kinA^E186K-GFP) as template. We then used two oligos, K5UTR2 and KinR1, for a fusion PCR to fuse the two fragments to generate a ∼2.1-kb fragment. We co-transformed the ∼2.1-kb fragment with the ∼4.6-kb kinA-GFP-AfpyrG-containing fragment (amplified using K5U2 and Kin3R as primers) into the XY42 strain. The transformants were screened by microscopically observing the GFP signals. In addition, we also performed a PCR using K5UTR3 and GFP5R2 as primers, and sequenced the PCR product with primers K5UTR2, KinR1, K5U3, and GFP5R2 to verify the E178K and E186K mutations to verify the E178K and E186K mutations in the absence of other mutations. However, the sequencing analysis detected the unintended K735R mutation.

Images were captured using an Olympus IX73 inverted fluorescence microscope linked to a PCO/Cooke Corporation Sensicam QE cooled CCD camera and controlled by the IPlab software. An UPlanSApo 100× objective lens (oil) with a 1.40 numerical aperture (NA) was used. A filter wheel system with GFP/mCherry-ET Sputtered series with high transmission (Biovision Technologies) was used. Images used for line scans and those for Fig. 2 E, Fig. 4, G–K, and Fig. S2 A were generated using a Nikon Ti2-E inverted microscope with Ti2-LAPP motorized Total Internal Reflection Fluorescence (TIRF) module and a Chromatic Aberration Free Infinity (CFI) apochromat TIRF 100 x 1.49 NA objective lens (oil). The microscope was controlled by NIS-Elements software using 488 and 561 nm lines of LUN-F laser engine and ORCA-Fusion BT cameras (Hamamatsu). For all images, cells were grown in the LabTek Chambered #1.0 borosilicate coverglass system (Nalge Nunc International). Images were taken at room temperature immediately after the cells were taken out of the incubators. Cells were cultured overnight in minimal medium with 1% glycerol and supplements at 32°C. The IPLab software (for the Olympus IX73 microscope) or the NIS-Elements software (for the Nikon Ti2-E microscope) was used for image acquisition and analysis. Image labeling was done using Microsoft PowerPoint and/or Adobe Photoshop. For quantitation of dynein comet signal intensity, a region of interest (ROI) was selected, and the IPLab program was used to measure the total signal intensity (Sum) within the ROI. The ROI box was then dragged to a nearby area outside of the cell to take the background value, which was then subtracted from the intensity value. For quantitation of HookA-GFP-marked early endosome accumulation at the hyphal tip, the total hyphal-tip signals (Sum) were measured, and background subtracted the same way. For quantitation of septal dynein signals, a region of interest (ROI) was selected and the IPLab program was used to measure the maximal signal intensity (Max) within the ROI. The ROI box was then dragged to a nearby area outside of the cell to take the background value, which was then subtracted from the intensity value. Hyphae were chosen randomly as long as the hyphal tip segment looked focused. For measuring the signal intensity of a microtubule plus-end comet formed by GFP-dynein, only those comets close to the hyphal tip were measured. For measuring GFP-dynein signal intensity at septa, usually only the septum most proximal to the hyphal tip was measured. For line scans, we draw a line starting from the hyphal tip in the middle of the hypha and get the average intensity value for the width of 2 μm, which is normally the hyphal width.

All statistical analyses were done using GraphPad Prism 9 for macOS (version 9.4.1, 2022). The D’Agostino & Pearson normality test was performed on all data sets (except for the percentage data presented in Fig. 3, E and H and Fig. S2 G where n is 3). For data sets that passed the normality test, t test (unpaired, two tailed) was used for analyzing two data sets and ordinary one-way ANOVA (unpaired) for multiple data sets. For data sets that did not pass the normality test, non-parametric tests were used without assuming Gaussian distribution. Specifically, Mann–Whitney test (unpaired, two tailed) was used for analyzing two data sets and the Kruskal–Wallis ANOVA test (unpaired) with Dunn’s multiple comparisons test was used for analyzing multiple data sets. Note that adjusted P values were generated from the Kruskal–Wallis ANOVA test with Dunn’s multiple comparisons test. For the percentage data presented in Fig. 3, E and H; and Fig. S2 G where n is 3, which is too small for the normality test, we used ordinary one-way ANOVA (unpaired) for statistical analysis. For analyzing the line scans of two groups, we used two-way ANOVA with Bonferroni’s multiple comparisons test to compare each cell mean with other cell mean in that row (Note that each row represents a position with a particular distance from the hyphal tip). For analyzing the line scans of multiple groups, we used two-way ANOVA with Tukey’s multiple comparisons test to compare each cell mean with other cell mean in that row.

Fig. S1 presents a protein sequence alignment of three different kinesin-1s. Fig. S2 shows phenotypes of the kinA^K895* mutant and various diploids. Video 1 shows microtubules labeled by GFP-TubA in a wild-type hypha. Video 2 shows microtubules labeled by GFP-TubA in a kinA^K895* mutant hypha. Table S1 lists A. nidulans strains used in this study.

We thank Berl Oakley, Miguel Peñalva, Bo Liu, Samara Reck-Peterson, Martin Egan, and Reinhard Fischer for Aspergillus strains, and the National Institutes of Health (initially by RO1 GM121850 to X. Xiang and currently by R35 GM140792 to X. Xiang) for funding.

Disclaimer: The opinions and assertions expressed herein are those of the author(s) and do not necessarily reflect the official policy or position of the Uniformed Services University or the Department of Defense.

Author contributions: R. Qiu and X. Xiang conceived the experiments. R. Qiu, J. Zhang and X. Xiang carried out the experiments. X. Xiang and R. Qiu wrote the manuscript, which was read and edited by all authors.