Biology subjects

Macfarlane, J. E.

Publications and source records attributed to Macfarlane, J. E..

3 recordsLinked to original sources

bioRxiv2026

Melanophilin, a Myosin Va Adapter Protein, Biases Track Selection of Myosin Va-and Kinesin-1-Transported Liposomes at Actin-Microtubule Intersections In Vitro

Secretory vesicle transport from the Golgi to the cell membrane involves kinesin and myosin Va motors on the vesicle surface cooperatively navigating their shared cargo through numerous actin-microtubule (MT) intersections. How the track on which the cargo exits the intersection is selected so that vesicles are delivered to their destination with spatial and temporal fidelity remains unclear. Here we hypothesized that melanophilin -- the adapter that links myosin Va to pigmented melanosomes and can bind to both actin and MTs -- acts as a phosphorylation-dependent switch to bias track preference at actin-MT intersections. To test this, we modeled melanosome transport in vitro using 350-nm liposomes with ~5 surface-bound molecules each of constitutively active myosin Va, kinesin-1, and full-length melanophilin with varying phosphorylation levels. Liposomes were then challenged with actin-MT intersections. Regardless of the track the liposomes entered the intersection on, liposomes with phosphorylated melanophilin were biased towards exiting the intersection on actin filaments while those with dephosphorylated melanophilin were biased to exit on MTs. Consistent with this, phosphorylated melanophilin showed a 2-fold preference to bind actin over MTs, and slowed liposome transport by myosin Va along actin filaments by ~40% by effectively acting as an anchor. Conversely, dephosphorylated melanophilin preferentially bound (2-fold) MTs over actin and, by acting as a tether, increased the kinesin-1 liposome transport distance on MTs. Therefore, melanophilin, based on its phosphorylation state, can bias track selection of cargo transported by kinesin-1 and myosin Va through the cell's complex cytoskeletal network with its numerous actin-MT intersections.

biophysics↗

bioRxiv2026

Cryo-EM Structure of Human ATAD2B Reveals a Hexameric Organization Contributes to ATPase Activity and Substrate Coordination

ATPase family AAA+ domain-containing protein 2B (ATAD2B) is a poorly characterized member of the ATAD2-like protein family, which contains a unique combination of tandem AAA+ ATPase domains with a C-terminal bromodomain. In humans, ATAD2B is dysregulated in several disease states including cancer and respiratory disorders, yet despite its promise as a therapeutic target, little is known about its molecular function. Here, we report the first high-resolution cryo-EM structure of human ATAD2B at 3.0 [A], revealing a two-tiered hexameric assembly with a shallow spiral staircase architecture. Structural analysis uncovers conserved AAA+ ATPase features, including nucleotide coordination at inter-subunit interfaces, inter-subunit signaling (ISS) gate loops, and pore loops that engage a substrate within the central channel. Biochemical assays demonstrate that ATAD2B is an active enzyme with an ATP hydrolysis rate of 0.34 ATP/hexamer/sec. Furthermore, the integrity of the hexameric complex is stabilized through unique knob-hole interactions, a linker arm that extends between the AAA2 and bromodomain, and an N-terminal linker domain (LD). These findings establish ATAD2B as a functional AAA+ ATPase and provide mechanistic insight into its enzymatic activities, laying the foundation for understanding its role in chromatin-associated processes.

biochemistry↗

bioRxiv2025

BicD and MAP7 collaborate to activate homodimeric Drosophila kinesin-1 by complementary mechanisms

The folded auto-inhibited state of kinesin-1 is stabilized by multiple weak interactions and binds weakly to microtubules. Here we investigate the extent to which homodimeric Drosophila kinesin-1 lacking light chains is activated by the dynein activating adaptor Drosophila BicD. We show that one or two kinesins can bind to the central region of BicD (CC2), a region distinct from that which binds dynein-dynactin (CC1) and cargo-adaptor proteins (CC3). Kinesin light chain significantly reduces the amount of kinesin bound to BicD and thus regulates this interaction. Binding of kinesin to BicD increases the number of motors bound to the microtubule, the fraction moving processively and the run length, suggesting that BicD relieves kinesin auto-inhibition. In contrast, microtubule-associated protein 7 (MAP7) has minimal impact on the percentage of motors moving processively but enhances both kinesin-1 recruitment to microtubules and run length. BicD relieves auto-inhibition of kinesin, while MAP7 enables activated motors to engage productively with microtubules. When BicD and MAP7 are combined, the most robust activation of kinesin-1 occurs, highlighting the crosstalk between adaptors and microtubule associated proteins in regulating transport. These observations imply that when both dynein and kinesin-1 are simultaneously bound to BicD, the direction the complex moves on MTs will be influenced by MAP7 and the number of bound kinesins.

biophysics↗