Lipid - Motor Interactions: Soap Opera or Symphony?

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ScienceDirect Lipid - Motor Interactions: Soap Opera or Symphony? Divya Pathak and Roop Mallik Intracellular transport of organelles can be driven by multiple motor proteins that bind to the lipid membrane of the organelle and work as a team. We review present knowledge on how lipids orchestrate the recruitment of motors to a membrane. Looking beyond recruitment, we also discuss how heterogeneity and local mechanical properties of the membrane may influence function of motor-teams. These issues gain importance because phagocytosed pathogens use lipid-centric strategies to manipulate motors and survive in host cells. Address Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400 005, India Corresponding author: Mallik, Roop ([email protected])

Current Opinion in Cell Biology 2016, 44:79–85 This review comes from a themed issue on Cell Architecture Edited by Terry Lechler and Rafael Carazo Salas For a complete overview see the Issue and the Editorial Available online 30th September 2016 http://dx.doi.org/10.1016/j.ceb.2016.09.005 0955-0674/# 2016 Elsevier Ltd. All rights reserved.

Introduction Motor proteins are ATPases that convert chemical energy into mechanical energy to drive many cellular functions including intracellular transport of vesicles [1] (Box 1). Vesicular transport entails cargo recognition by the respective motor, transport and possible cargo-motor dissociation at destination. Recognition of a cargo by motor(s) can be mediated by heterogeneities in protein and lipid composition of the cargo. Each subcellular organelle has a characteristic set of membrane lipids that encloses its inner contents within a bilayer membrane (Figure 1). This membrane is the substrate on which motors must attach (directly or indirectly) before they can effect transport. Lipid composition and local membrane heterogeneity are therefore important for recruiting motors, and perhaps also in deciding the micro-organization of motors on the cargo surface. However, the role of lipids is less appreciated in the literature that discusses regulation of motors on a cargo. Given the vast variety of cellular vesicles, their lipids and their motors, reliable mechanisms must ensure cargo-motor recognition. This requires a set of diverse protein domains to be matched to another set of even more diverse lipids on the membrane. Is this matching just[1_TD$IF] a television soap opera[5_TD$IF], where characters www.sciencedirect.com

meet each other transiently to develop random intimacy/ [6_TD$IF]dislike, or is it[1_TD$IF] a well-orchestrated symphony where precise rules dictate matchmaking in a spatio-temporally controlled manner? The lipid bilayer membrane on many organelles largely contains phospholipids along with some sphingolipids and cholesterol (Box 2). These lipids can affect membranebound proteins through their hydrophilic head group or hydrophobic fatty acid chain. For example, the antibiotic Gramicidin A that functions as a cation channel in lipid membranes is rendered non-functional upon conditions of hydrophobic mismatch with the phospholipid carbon chains [2]. Proteins contain different domains that allow them to interact with either specific lipid headgroups or to sense the overall membrane curvature. There are atleast 12 different types of phospholipid binding domains in proteins (Figure 1) that govern their activity and localization on lipid membranes [3]. The most common is the PH (pleckstrin homology) domain, made up of 120 amino acids that binds to PI(4,5)P (phosphatidyl inositol-4,5bisphosphate) [3,4]. Phospholipids are not the only lipid moieties that are sensed by proteins. Proteins belonging to OSBP (oxysterol binding protein) families contain a domain that can bind oxysterols and sterols [5]. The effect of such sterol sensing proteins on intracellular transport is exemplified by ORP1L, which contains a sterol sensing ORD domain that regulates motion of late endosomes through a tripartite complex containing Rab7, RILP and dynein-dynactin [6]. A large body of work has identified adaptor proteins for motor recruitment to cargoes [7,8,9]. It is also realized that most cellular cargoes can interact with different motor classes. For example, the miro/milton complex can interact with microtubule motors dynein and kinesin to mediate bidirectional transport of mitochondria [10,11]. Similarly, JIP1 (JNK interacting protein), a scaffolding protein present on autophagosomes can regulate its interaction with dynein and kinesin-1, depending on its phosphorylation status [8]. We shall not discuss these issues here, and refer the reader to reviews on motor-cargo recognition [9] and bidirectional transport for more information [12,13]. This review addresses microtubule-based motors and their recruitment onto vesicular cargo, directly or indirectly determined by the lipid composition of cargo membrane.

Lipids confer identity to cellular organelles The membrane of different subcellular organelles varies in lipid composition, and this compartmentalization of different lipids can mediate protein recruitment via Current Opinion in Cell Biology 2017, 44:79–85

80 Cell Architecture

Box 1 MOTOR PROTEINS Intracellular transport occurs on actin and microtubule filaments. Actin based transport is carried out by the myosin class of motors over shorter distances, whereas microtubule motors like kinesin and dynein mediate long-range transport [1]. Myosins have a conserved domain organization with a motor domain at the N-terminus, a neck region that can bind myosin light chains or calmodulin and a variable tail domain that can interact with cargo. The tail domain can target myosin to specific cargos. Some myosins, like class I and IX, are monomers whereas class II and V exist as dimers. An alpha helical coiled coil region determines the dimerization capability. Myosins and kinesins belong to a superfamily of G-proteins whereas dynein belongs to AAA ATPase superfamily.

Myosin-V

Cytoplasmic dynein

Kinesin-1 Light chains

Tail domain

IC Tail

Tetex-1 LC8 Roadblock

Light chains

LIC Heavy Chains

Heavy Chains

Linker

Light Chains Motor Domain

Motor Domain Motor Domain Stalk Current Opinion in Cell Biology

Cytoskeletal motors: Myosin-V consists of a dimer of myosin heavy chain, each of which associates with 6 myosin light chains. Conventional kinesin (kinesin-1) is a dimer of two kinesin heavy chains and two kinesin light chains. Cytoplasmic dynein is composed of two each of dynein heavy chains, intermediate chains, light intermediate chains and three light chains. Conventional kinesin (kinesin-1) is a heterotetramer containing two kinesin heavy chains (KHCs) and two kinesin light chains (KLCs). KHC consists of an N-terminal motor domain, neck domain, a coiled coil stalk and a C-terminal tail domain. The neck linker allows for processive motion of the motor and mediates dimerization. The tail domain plays a role in binding cargo. KLC contains a heptad repeat region that mediates interaction with KHC, and TPR motifs (tetratricopeptide repeat) that bind cellular cargos. Kinesin-2 is a predominantly heterotrimeric motor that contains two heavy chains and one accessory protein called KAP (kinesin associated protein). Kinesin-3, which exists as a monomeric motor in mammalian cells, has been shown to dimerize on the cargo resulting in a processive motor [32]. Dynein is an approximately 1.4 MDa multi protein complex, which consists of atleast 8 polypeptides that play a role in cargo binding and in the the mechanochemical cycle of dynein [1]. The largest polypeptide - dynein heavy chain (DHC) is approximately 500 kDa and contains the motor domain which binds microtubules and ATP. The motor domain is quite complex and contains a ring of six AAA+ domains, of which AAA1 is the main site for ATP hydrolysis. The microtubule-binding domain emerges from AAA4 region and can change its microtubule affinity depending on ATP or ADP bound state. The linker, located N terminus to the AAA ring, undergoes conformational change during dynein’s powerstroke and is important for motility. Dynein has the ability to shorten its stepsize in response to opposing load [49], and this could be a reason why dynein functions well in large teams, whereas kinesin does not [43]. The assembly of dynein-teams on cholesterol rich membrane domains [21] becomes important in this background.

lipid-protein interaction (Figure 1). In other words, the organelle’s lipidome and proteome are intimately related. Since phosphoinositides comprise <10% of total cellular lipids, and can be differently phosphorylated, they can serve as a lipid code to establish organelle identity [14]. As an example, PI(4,5)P is mainly present at the plasma membrane but PI(4)P is highly enriched in the Golgi. Phosphatidylinositol-3-phosphate [PI(3)P], which determines the identity of early endosome compartments, can recruit effector proteins containing FYVE or PX domains like EEA1 and is essential for endosome/phagosome maturation [15]. During maturation of early endosomes/phagosome PI(3)P may get dephosphorylated to phosphatidylinositol, or it may get phosphorylated to form phosphatidylinositol-(3,5)phosphate [PI(3,5)P] [16,17,18]. Phosphoinositides are therefore compartmentalized with respect to different Current Opinion in Cell Biology 2017, 44:79–85

stages of the endosomal pathway. Addition/removal of phosphates to lipid moieties could therefore modulate motor recruitment to an organelle along specific cellular pathways. As noted earlier, cholesterol could determine motor recruitment to a cargo. The cholesterol amount is highly variable among subcellular organelles, with the ER containing only 1% of total cellular cholesterol whereas the plasma membrane has 50-80% [19]. Intermediate levels of cholesterol are present in Golgi and endocytic compartments. Cholesterol and sphingolipids lead to formation of lipid microdomains/lipid rafts in the plasma membrane, which are much debated dynamic entities varying from 20 nm to 200 nm [20]. We have recently observed cholesterol enrichment on phagosome membranes as a function of phagosome maturation. This www.sciencedirect.com

Lipid - Motor Interactions: Soap Opera or Symphony? Pathak and Mallik 81

Figure 1

Dynein-Dynactin complex p62 p

1

Dynein Dynactin complex

AnkyrinB y

6

p62 AnkB

Unc104/KIF1A (Kinesin-3)

PI(3)P

Unc104/KIF1A (Kinesin-3)

PI(4,5)P KIF16B (Kinesin-3)

PI(3)P

2

KIF16B (Kinesin-3) Arp1 Spectrin

5

DyneinDynactin n complex

Kinesin-1

Spectrin

Phosphatidic Acid (PA) Cholesterol

PA

ORP1L O

Phosphatidylinositol-4,5bisphosphate [PI(4,5)P] SNX

?

Rab Rab7 7

PI(3) or

1L

ORP

Rab

7 Rab

RILP

Sphingolipids

RILP

RILP

PI(3,5)P + Curvature

4

Sorting ti Nexins

? DyneinKinesin Dynactin complex

3

DyneinDynactin complex

Phosphatidylinositol-3phosphate [PI(3)P] Phosphatidylinositol-3phosphate [PI(3)P] or Phosphatidylinositol-3,5bisphosphate [PI(3,5)P]

Cholesterol Current Opinion in Cell Biology

Lipids lead the way. Scheme illustrates how different membrane lipids mediate motor interaction. (1) PI(3)P can associate with dynein-dynactin complex via ankyrinB. (2) PI(3)P can also interact directly with KIF16B. (3) Cholesterol sensor (ORP1L) associates with dynein-dynactin complex through Rab7-RILP and can help to cluster many dyneins into a microdomain/raft. (4) Sorting nexins (SNX) that sense phosphatidylinositides and membrane curvature may mediate dynein or kinesin driven motion. (5) Acidic phospholipids like phosphatidic acid can recruit dynein-dynactin complex via spectrin. (6) Unc 104 can directly bind to PI(4,5)P using its PH domain.

induces formation of lipid rafts on the late phagosome membrane, where teams of dynein motors are then assembled to drive rapid transport of the phagosome towards perinuclear lysosomes [21[2_TD$IF],22]. Such a mechanism could also be true for endosomes and autophagosomes. Recruitment of motors via cholesterol could therefore modulate motor-cargo interactions and localize endophagocytic compartments to different cellular locations as they mature [23,24].

Direct and Indirect Coupling of Motors to Lipid membranes Motors could get recruited to a cargo by directly binding to lipids, or via intermediate adaptor proteins that can bind the lipid membrane. Initial studies with cytoplasmic dynein showed that this motor could bind phospholipid vesicles, and binding stimulated dynein’s ATPase activity even in the absence of microtubules [25,26]. Remarkably, www.sciencedirect.com

simple incorporation of acidic phospholipids (e.g. PA) into protein-free liposomes is sufficient to recruit the dyneindynactin complex from squid axoplasm[8_TD$IF], and to drive in vitro liposome motion comparable to axonal vesicles [27[7_TD$IF]]. The kinesin-3 family motor Unc104 binds to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P] on synaptic vesicles through its PH domain [28,29]. KIF16B, another member of kinesin-3 family, has a PX domain which interacts with phosphatidylinositol-3-phosphate [PI(3)P], as demonstrated by in vitro motion of liposomes. PI(3)P on early endosomes also associates with KIF16B in vivo [30]. Binding to lipids can also activate motors. The monomeric non-processive Unc104 kinesin dimerizes on the cargo membrane to convert to a super processive motor, possibly through PI(4,5)P and PI(3)P enriched microdomains [28,29,31,32,33]. Similarly, the minus end directed kinesin 14-VIb from the moss Physcomitrella patens, which Current Opinion in Cell Biology 2017, 44:79–85

82 Cell Architecture

Box 2 LIPIDS AND THE MEMBRANES THEY MAKE Biophysical properties of lipids are determined by the nature of their acyl chain and head group. The length and degree of saturation of the acyl chain determines the thickness and packing of the lipid bilayer. Membrane lipids consist of phospholipids, sphingolipids and cholesterol. Phospholipids are characterized by the differences in their head groups, which plays an important role in lipid-protein interaction. The predominant phospholipids in the lipid membrane are phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidic acid (PA). The two major physical properties of the membrane are its curvature and fluidity.

LPC, PIPs (Positive)

PC, PS (Cylindrical)

DAG, PA (Negative)

Current Opinion in Cell Biology

The curvature of a lipid membrane is primarily determined by the comparative sizes of the head group and the fatty acid chain of constituent lipids. PC, PS and Sphingomyelin are cylindrical and therefore make planar bilayers, Lipids in which the head group is larger (e.g. phosphoinositides and lysophospholipids) form a positive curvature resulting in formation of micellar structures. Lipids which have a smaller head groups (e.g. diacylglycerol and PE) induce negative curvature in a membrane. The fluidity of the membrane is governed by the saturation of the fatty acyl chains. The membrane can exist in three phases - solid/gel phase, liquid ordered (Lo) phase and liquid disordered (Ld) phase. The Lo phase, which mainly consists of unsaturated fatty acyl chains like oleic acid, has higher fluidity. The Lo phase that exhibits reduced fluidity may contain cholesterol, sphingolipids and PC (mostly containing saturated fatty acyl chain). Cholesterol intercalates preferably with sphingolipids to shield itself from the aqueous environment that results in formation of lipid microdomains/lipid rafts, as demonstrated through decreased diffusion within these domains.

is non processive as native dimers, becomes processive when artificially clustered on liposomes [34]. Since plants do not have cytoplasmic dynein, clustering of this kinesin may be a means to achieve processive retrograde motion. Apart from the above scenario of direct lipid-motor interaction, motors can also be recruited via intermediate adaptor proteins that associate with specific lipids. Sorting nexins, which associate with distinct cellular compartments, contain two membrane binding regions - a BAR domain that senses curvature, and a PX domain that recognizes phosphoinositides [3]. Sorting nexins associate with and mediate transport of different endocytic compartments. The PX domain of SNX1 and SNX2 can interact with endosomal PI(3)P and PI(3,5)P. SNX1 is a part of the retromer complex that mediates transport from early endosomes to the trans-Golgi network. The retromer complex also includes SNX5 or SNX6, which can interact with p150 of dynein-dynactin complex [35]. PI(3)P mediates retrograde axonal transport for a variety of neuronal cargos by recruiting ankyrinB, which in turn binds p62 of dynein-dynactin complex [36]. AnkyrinB, which binds to PI(3)P, has been speculated to target Current Opinion in Cell Biology 2017, 44:79–85

proteins to membrane domains [37]. Apart from membrane recruitment, lipids may also dissociate motors from cargo, as has been observed for PI(4)P mediated dissociation of dynein from the trans-Golgi membrane [38]. Phosphatidylinositol 4,5-bisphosphate [10_TD$IF](PIP2[1_TD$IF]) dependent recruitment of dynein to the cell cortex is important for positioning the mitotic spindle and progression of mitosis [39].

Heterogeneity and Mechanical properties and membranes: Implications for Cargo transport and Pathogen biology Setting aside the details of above stated interactions, one would also like to understand general physical principles behind motor-membrane recognition. A combination of electrostatics, lipid-packing defects and local curvature delineates membrane territories inside cells [40]. Charged lipids [e.g. PS and PI(4,5)P] are more abundant in the late secretory pathway (e.g. endosomes and PM) compared to the early secretory pathway (e.g. the ER and cis-Golgi). As stated above, both kinesin and dynein can bind acidic phospholipids. Electrostatics may therefore play an important role in motor-cargo recognition within different membrane compartments, but much of this is unknown. www.sciencedirect.com

Lipid - Motor Interactions: Soap Opera or Symphony? Pathak and Mallik 83

Figure 2

Cholesterol rich platform

Phagosome Dynein

(+)

Phagosome

Increase in

Clustering of

Maturation

Cholesterol

Dynein

( –)

Microtubule

Early Phagosome

Team of Dyneins

Late Phagosome

Lysosome Current Opinion in Cell Biology

Clustering of Dynein on phagosomes drives phagosome-lysosome fusion. Pathogens are usually killed inside the macrophage cells of our immune system. Macrophage cells enclose the pathogen within a phagosome. The phagosome is then carried by motors to the acidic lysosome. Phagosomes initially move in back-and-forth manner (double headed arrow on early phagosome), but then switch to more unidirectional motion (arrow on late phagosome) which rapidly takes them towards the lysosome for degradation. This figure depicts that the switch is caused by clustering of dynein into cholesterol rich platforms/microdomains (also called lipid rafts) on the membrane of a phagosome. One such dynein-team at the bottom of the late phagosome is rapidly transporting it (and the pathogen inside) towards the lysosome. Other motors (e.g. kinesin) are not shown for the sake of simplicity.

In an opposite trend to electrostatics, lipids are better packed in membranes of the late secretory pathway, but packing defects increase in membranes of the early secretory pathway. These defects play a major role in binding of proteins (and therefore possibly motors) to the membrane [40]. This brings us to the role of cholesterol, which improves lipid packing by preferentially stacking with saturated (straight-chain) lipids. It is being increasingly recognized that motors work as a team in various cellular functions including transport [41,42,43,44]. We have recently demonstrated that cholesterol enrichment leads to geometric clustering of dyneins into cholesterol-rich microdomains on late phagosomes [21[9_TD$IF]]. This assembles a dynein-team within a microdomain, allowing many dyneins to simultaneously engage a single microtubule and generate cooperative force that shifts the transport of phagosomes from bidirectional to retrograde (see schematic in Figure 2). Most importantly, a glycolipid (lipophosphoglycans; LPG) purified from the pathogenic parasite Leishmania Donovani inhibits clustering of dynein and the resultant retrograde motion. The observed assembly of motor-teams on cholesterol rich domains [21,22] suggests the exciting possibility that a mechanically stiff substrate with low packing defects is used for efficient force generation by the dyneins. When thinking of the cooperative function of many motors clustered into a cholesterol-rich microdomain, one must remember that protein-lipid interactions www.sciencedirect.com

are not bimolecular because a lipid molecule is much smaller than a protein [40]. Therefore, a dynein motor (or its adaptor) likely binds to the membrane via multiple weak interactions with many lipid moieties. Perhaps a membrane that is mechanically rigid on those length scales is also favorable for single as well as cooperative dynein function. Molecules that sense force/tension (e.g. integrins, cadherins, mechanosensitive channels) associate preferentially with cholesterol rich domains [45]. It is therefore not so surprising if force-generating molecule (e.g. the dynein motor) also takes advantage of membrane rigidity within cholesterol-rich domains. While the above examples suggest that some motors work better on an ordered microdomain, somewhat opposite behavior is seen when myosin-Va motors are coupled to a liposome membrane [46]. Velocity of multiple-myosin ensembles on fluid-phase DOPC liposomes is significantly higher than single myosins. It was suggested that leading and trailing myosins respectively experience resistive and assistive loads. Trailing myosins therefore detach preferentially from the MT and the vesicle recenters on the leading motor to enhance cargo velocity. This asymmetry of detachment is lost for myosins on more ordered membranes (as in DPPC liposomes), and the velocity enhancement is not seen. It therefore appears that single-motor function and membrane rigidity are both important in deciding the ensemble function of motors on a lipid membrane. Current Opinion in Cell Biology 2017, 44:79–85

84 Cell Architecture

Conclusions and Future Perspectives Lipids are not inert molecules that just segregate intracellular compartments, but rather they play multiple roles ranging from signaling to intracellular trafficking. Given the diversity of lipid molecules, we have only just begun to understand how membrane lipids influence membrane-motor interaction and function. Many pathogens alter membrane lipid microdomains [18,47] and may ‘‘hijack’’ motors to enter and survive in the host cell [48]. Lipid-centric immune evasion strategies of pathogens can disrupt the stereotypic spatio-temporal localization of phagosomes that helps degrade pathogens inside macrophage cells. This suggests that pathogens can manipulate motor-lipid interactions to their advantage. There is a particularly pressing need to address these questions. Cell biologists/Biophysicists often do not have the background knowledge, permissions and facilities required for pathogen research. Much could be learnt through collaborations with pathogen biologists.

Acknowledgements RM acknowledges funding through an International Senior Research Fellowship from the Wellcome Trust UK (grant WT079214MA), and also a Wellcome Trust–Department of Biotechnology Senior Fellowship (grant IA/S/11/2500255). We thank Ashim Rai for comments and discussion. Our apologies to colleagues whose work we could not cite for reasons of space.

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Current Opinion in Cell Biology 2017, 44:79–85