Irina Semenova

CLIP-170-Dependent Capture of Membrane Organelles by Microtubules Initiates Minus-End Directed Transport

Cytoplasmic microtubules (MTs) continuously grow and shorten at free plus ends. During mitosis, this dynamic behavior allows MTs to capture chromosomes to initiate their movement to the spindle poles; however, the role of MT dynamics in capturing organelles for transport in interphase cells has not been demonstrated. Here we use Xenopus melanophores to test the hypothesis that MT dynamics significantly contribute to the efficiency of MT minus-end directed transport of membrane organelles. We demonstrate that initiation of transport of membrane-bounded melanosomes (pigment granules) to the cell center involves their capture by MT plus ends, and that inhibition of MT dynamics or loss of the MT plus-end tracking protein CLIP-170 from MT tips dramatically inhibits pigment aggregation. We conclude that MT dynamics are required for the initiation of MT transport of membrane organelles in interphase cells, and that +TIPs such as CLIP-170 play an important role in this process.

CK1 activates minus-end–directed transport of membrane organelles along microtubules

This study shows that the signal transduction pathway responsible for the initiation of minus-end–directed movement of membrane-bounded pigment granules in melanophores involves sequential activation of protein phosphatase 2A and casein kinase 1 and that this activation correlates with increased phosphorylation of the dynein intermediate chain.

Switching of membrane organelles between cytoskeletal transport systems is determined by regulation of the microtubule-based transport

Intracellular transport of membrane organelles occurs along microtubules (MTs) and actin filaments (AFs). Although transport along each type of the cytoskeletal tracks is well characterized, the switching between the two types of transport is poorly understood because it cannot be observed directly in living cells. To gain insight into the regulation of the switching of membrane organelles between the two major transport systems, we developed a novel approach that combines live cell imaging with computational modeling. Using this approach, we measured the parameters that determine how fast membrane organelles switch back and forth between MTs and AFs (the switching rate constants) and compared these parameters during different signaling states. We show that regulation involves a major change in a single parameter: the transferring rate from AFs onto MTs. This result suggests that MT transport is the defining factor whose regulation determines the choice of the cytoskeletal tracks during the transport of membrane organelles.

Cytoplasmic Dynein is Involved in the Retention of Microtubules at the Centrosome in Interphase Cells

Cytoplasmic dynein is known to be involved in the establishment of radial microtubule (MT) arrays. During mitosis, dynein activity is required for tethering of the MTs at the spindle poles. In interphase cells, dynein inhibitors induce loss of radial MT organization; however, the exact role of dynein in the maintenance of MT arrays is unclear. Here, we examined the effect of dynein inhibitors on MT distribution and the centrosome protein composition in cultured fibroblasts. We found that while these inhibitors induced rapid (t1/2 ∼ 20 min) loss of radial MT organization, the levels of key centrosomal proteins or the rates of MT nucleation did not change significantly in dynein‐inhibited cells, suggesting that the loss of dynein activity does not affect the structural integrity of the centrosome or its capacity to nucleate MTs. Live observations of the centrosomal activity showed that dynein inhibition enhanced the detachment of MTs from the centrosome. We conclude that the primary role of dynein in the maintenance of a radial MT array in interphase cells consists of retention of MTs at the centrosome and hypothesize that dynein has a role in the MT retention, separate from the delivery to the centrosome of MT‐anchoring proteins.

Regulation of microtubule-based transport by MAP4

Binding to microtubules of Xenopus microtubule-associated protein 4 (XMAP4) negatively regulates dynein-dependent movement of membrane organelles and positively regulates kinesin-2–based movement. Phosphorylation reduces binding of XMAP4 to microtubules and therefore regulates the direction of microtubule-based transport.

The protein kinase A-anchoring protein moesin is bound to pigment granules in melanophores.

Major signaling cascades have been shown to play a role in the regulation of intracellular transport of organelles. In Xenopus melanophores, aggregation and dispersion of pigment granules are regulated by the second messenger cyclic AMP through the protein kinase A (PKA) signaling pathway. PKA is bound to pigment granules where it forms complexes with molecular motors involved in pigment transport. Association of PKA with pigment granules occurs through binding to A‐kinase‐anchoring proteins (AKAPs), whose identity remains largely unknown. In this study, we used mass spectrometry to examine an 80 kDa AKAP detected in preparations of purified pigment granules. We found that tryptic digests of granule protein fractions enriched in the 80 kDa AKAP contained peptides that corresponded to the actin‐binding protein moesin, which has been shown to function as an AKAP in mammalian cells. We also found that recombinant Xenopus moesin interacted with PKA in vitro, copurified with pigment granules and bound to pigment granules in cells. Overexpression in melanophores of a mutant moesin lacking conserved PKA‐binding domain did not affect aggregation of pigment granules but partially inhibited their dispersion. We conclude that Xenopus moesin is an AKAP whose PKA‐scaffolding activity plays a role in the regulation of pigment dispersion in Xenopus melanophores.

Melanophores for Microtubule Dynamics and Motility Assays

Microtubules (MTs) are cytoskeletal structures essential for cell division, locomotion, intracellular transport, and spatial organization of the cytoplasm. In most interphase cells, MTs are organized into a polarized radial array with minus-ends clustered at the centrosome and plus-ends extended to the cell periphery. This array directs transport of organelles driven by MT-based motor proteins that specifically move either to plus- or to minus-ends. Along with using MTs as tracks for cargo, motor proteins can organize MTs into a radial array in the absence of the centrosome. Transport of organelles and motor-dependent radial organization of MTs require MT dynamics, continuous addition and loss of tubulin subunits at minus- and plus-ends. A unique experimental system for studying the role of MT dynamics in these processes is the melanophore, which provides a useful tool for imaging of both dynamic MTs and moving membrane organelles. Melanophores are filled with pigment granules that are synchronously transported by motor proteins in response to hormonal stimuli. The flat shape of the cell and the radial organization of MTs facilitate imaging of dynamic MT plus-ends and monitoring of their interaction with membrane organelles. Microsurgically produced cytoplasmic fragments of melanophores are used to study the centrosome-independent rearrangement of MTs into a radial array. Here we describe the experimental approaches to study the role of MT dynamics in intracellular transport and centrosome-independent MT organization in melanophores. We focus on the preparation of cell cultures, microsurgery and microinjection, fluorescence labeling, and live imaging of MTs.

Stimulation of the CLIP-170--dependent capture of membrane organelles by microtubules through fine tuning of microtubule assembly dynamics.

A combination of experimental and computational approaches shows that signals that stimulate minus end–directed transport of membrane organelles along microtubules change microtubule assembly dynamics in a way that enhances organelle capture by the growing microtubule tips.

Engineered Tug-of-War Between Kinesin and Dynein Controls Direction of Microtubule Based Transport In Vivo.

Bidirectional transport of membrane organelles along microtubules (MTs) is driven by plus‐end directed kinesins and minus‐end directed dynein bound to the same cargo. Activities of opposing MT motors produce bidirectional movement of membrane organelles and cytoplasmic particles along MT transport tracks. Directionality of MT‐based transport might be controlled by a protein complex that determines which motor type is active at any given moment of time, or determined by the outcome of a tug‐of‐war between MT motors dragging cargo organelles in opposite directions. However, evidence in support of each mechanisms of regulation is based mostly on the results of theoretical analyses or indirect experimental data. Here, we test whether the direction of movement of membrane organelles in vivo can be controlled by the tug‐of‐war between opposing MT motors alone, by attaching a large number of kinesin‐1 motors to organelles transported by dynein to minus‐ends of MTs. We find that recruitment of kinesin significantly reduces the length and velocity of minus‐end‐directed dynein‐dependent MT runs, leading to a reversal of the overall direction of dynein‐driven organelles in vivo. Therefore, in the absence of external regulators tug‐of‐war between opposing MT motors alone is sufficient to determine the directionality of MT transport in vivo.

Stimulation of microtubule-based transport by nucleation of microtubules on pigment granules

In Xenopus melanophores, nucleation of microtubules on pigment granules provides a positive feedback loop that enhances their transport to the cell center during pigment aggregation.