BICD2, dynactin, and LIS1 cooperate in regulating dynein recruitment to cellular structures

Abstract

This study dissects the recruitment of dynein and dynactin to cargo by a conserved motor adaptor BICD2. It is shown that dynein, dynactin, and BICD2 form a triple complex in vitro and in vivo. Investigation of the properties of this complex by direct visualization of dynein in live cells shows that BICD2-induced dynein transport requires LIS1.

Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility

“…To address this question, we purified an N‐terminal fragment of the human cargo adaptor protein BicD2 (BicD2‐N) (Fig EV1B). This fragment lacks the ability of autoinhibition (Hoogenraad et al , 2001) and has been shown to trigger processive dynein/dynactin motility (Splinter et al , 2012; McKenney et al , 2014; Schlager et al , 2014). Addition of 200 nM Alexa647‐labelled SNAP‐tagged BicD2‐N (Alexa647‐BicD2‐N) to a reconstitution assay with dynamic microtubules, dynein, dynactin and EB1 leads now to the appearance of processive and lattice‐bound GFP‐dyneins in addition to plus‐end accumulated dynein (Fig 2A).…”

“…In metazoan cells, BicD2 and dynactin cooperate with yet another regulator, the Lis1 protein, to control both processive motion as well as end tracking of dynein (Splinter et al , 2012). To investigate the mechanism of the combinatorial interplay of these different dynein regulators, we purified a fluorescent Lis1 construct (Fig EV1B) and combined all purified regulators in our dynamic microtubule end tracking assay.…”

“…Although 5 μM BicD2‐N strongly reduced microtubule end tracking of dynein (Fig 5A, and as shown earlier in Fig 2D, Movie EV2), combining 1 μM or 5 μM Lis1 with EB1, GFP‐dynein, dynactin and BicD2‐N showed that Lis1 can restore end tracking of GFP‐dynein in a dose‐dependent manner (Fig 5B and C, Movies EV5 and EV6), as quantitatively shown by the GFP‐dynein fluorescence profiles at growing microtubule plus ends (Fig 5D). This activity of Lis1 can explain the requirement of Lis1 for end tracking of dynein in cells, where the presence of several cargo adaptors, including BicD2, may suppress EB‐dependent end tracking of dynein (Coquelle et al , 2002; Splinter et al , 2012). …”

“…However, since BicD2‐N was not observed to track growing microtubule ends in the presence of end tracking dynein/dynactin, we consider it more likely that the DDB complex is in a conformational state that is less likely to interact with EB1 than with the microtubule directly. This scenario suggests a potentially simple competition‐based explanation for why overexpression of the BicD2‐N fragment in human cells abolished plus‐end tracking of dynein (Splinter et al , 2012). A similar mechanism for down‐regulating dynein end tracking by adapter proteins appears to exist in budding yeast where the expression of the cortical adaptor Num1 also removed dynein from microtubule plus ends (Lammers & Markus, 2015).…”

“…Lis1 has been reported to regulate both initiation of minus‐end‐directed motility (Egan et al , 2012; Splinter et al , 2012; Moughamian et al , 2013) and plus‐end tracking of dynein (Coquelle et al , 2002; Splinter et al , 2012) in cells. However, Lis1 was not required for dynein end tracking in a minimal in vitro reconstitution (Duellberg et al , 2014), raising the question as to why Lis1 is needed for dynein end tracking in cells.…”