Kinesin-mediated transport in the secretion of extracellular vesicles

Introduction

Intercellular communication is vital for maintaining cellular function and homeostasis. Extracellular vesicles (EVs) have emerged as a crucial mechanism of cell-to-cell communication facilitating interactions between neighboring cells or over long distances via the bloodstream or lymphatic system [1, 2].

EVs are a heterogeneous group of membrane-limited vesicles containing lipids, proteins, metabolites or nucleic acids, reflecting the genomic and proteomic content of their cell of origin [3]. Originally discovered as an alternative waste disposal mechanism, EVs are now accepted as an integral part of intercellular communication pathways that are involved in physiological as well as pathological conditions. EV secretion is highly conserved among species and has been reported in bacteria, protozoa, insects, plants and mammals [2, 4].

Two major classes of EVs can be distinguished: large EVs (lEVs) and small EVs (sEVs) also called exosomes. LEVs are commonly characterized as approximately up to 1 μm sized vesicles directly budding from the plasma membrane or membrane extrusions such as cilia, filopodia or microvilli and include microvesicles and apoptotic bodies. SEVs or exosomes, on the other hand, are commonly characterized as approximately 30–150 nm small vesicles of endosomal origin [5, 6, 7]. Another less frequently mentioned type of EVs are large oncosomes, which represent a group of approximately 1–10 μm large cancer-derived EVs [8].

Endocytic trafficking starts with the internalization of extracellular substances such as membrane receptors, ligands, nutrients, fluids, or membrane lipids. This process leads to the formation of an endocytic vesicle that is excised from the plasma membrane and taken up into the cytosol. Internalized vesicles can be transported to various intracellular compartments or they are recycled back to the plasma membrane [9]. Within the endosomal pathway, endocytic vesicles can directly fuse with an early endosome (EE), which can either develop into recycling endosomes (REs) or mature into late endosomes (LEs). LEs eventually develop into multivesicular bodies (MVBs), which evolve by a second invagination process that results in the inward invagination of the endosome limiting membrane for the formation of intraluminal vesicles (ILVs). During this process, cellular content destined for either degradation or exocytosis is sorted in the developing ILVs. Lastly, LEs can undergo fusion with lysosomes or autophagosomes for degradation or fusion with the plasma membrane, which results in the ILVs being released as exosomes into the extracellular space [6, 7, 10].

Despite significant progress, many questions about the secretion of sEVs remain unanswered. For instance, the underlying network governing cellular communication is still unknown and the mechanisms controlling secretory vs. degradative transport, as well as the key players of secretory transport remain elusive. This review will focus on the transport of endosomes through the cell and the involvement of motor proteins in this process.

Motor proteins

Motor proteins play a crucial role in intracellular transport, facilitating the movement of synthesized proteins, organelles, and other cellular components along cytoskeleton filaments to their final destination. Here, we will focus on kinesins and, to a minor extent, on cytoplasmic dynein molecular motors, both of which move along the microtubule tracks (MT) of the cell. Kinesins hereby commonly facilitate MT-plus-end-directed anterograde transport towards the cell periphery, whereas dyneins facilitate MT-minus-end-directed retrograde transport towards the microtubule-organizing center (MTOC). Kinesins belong to the kinesin superfamily of proteins (KIFs), a large family encoded by 45 mammalian genes, which also produce various isoforms through alternative splicing [11]. These proteins serve crucial functions in numerous cellular processes, including cell motility, cell division, intracellular transport of different cargos or organelles, and the regulation of the microtubule skeleton [12]. A typical kinesin motor comprises a conserved motor domain, along with distinct stalk and tail domains responsible for cargo binding, interaction with adaptors or scaffolds, and dimerization of KIFs. The movement of kinesins is driven by ATP binding and hydrolysis [11]. Transport processes along microtubules are regulated on multiple levels, including the activation or deactivation of motors, selective cargo binding, involvement of microtubule-associated proteins (MAPs) or post-translational modifications of MT-tracks [13, 14, 15, 16]. These regulatory mechanisms ensure the precise and efficient transport of cellular components within the cell.

Since this review will focus mostly on kinesins, we will give a short overview of the different members of this group of motor proteins. In mammals, there are 45 genes that encode various kinesins, which can be classified into 15 different families. Moreover, kinesins can also be grouped according to the relative position of their motor domain in N-, C-, and M-Kinesins. N-Kinesins, the largest group, facilitate plus-end directed anterograde transport, while C-Kinesins facilitate minus-end directed retrograde transport. M-Kinesins, on the other hand, are responsible for MT depolymerization [11]. The kinesins associated with endosomal compartment transport predominantly belong to the kinesin-1, kinesin-2, and kinesin-3 family. The kinesin-1 family comprises the members Kif5A, Kif5B, and Kif5C, which homodimerize and assemble into tetrameric complexes with two kinesin light chains. Kif5A and Kif5B are exclusively expressed in neurons, while Kif5C is ubiquitously expressed [17]. The kinesin-2 family includes Kif3A, Kif3B, Kif3C, and Kif17. Whilst Kif3A, Kif3B, and Kif3C form heterotrimeric complexes comprising two motor domains and an accessory domain, Kif17 only forms homodimers [17]. The kinesin-3 family includes Kif1A, Kif1B, Kif1C, Kif13A, Kif13B, Kif14, Kif16A and Kif16B. Once these members undergo homodimerization, they exhibit fast processive movement, which is an ideal prerequisite for long-range transport processes [18, 19].

Indeed, many studies suggest the involvement of kinesins in the anterograde transport of endosomal compartments leading to either the secretion of ILVs to the extracellular space or the fusion of endosomal compartments with the lysosome for content degradation. Thus, the following section will further illustrate how kinesins engage in the transport of endosomal compartments.

Kinesins in endosomal transport processes

The maturation of endosomal compartments from early to late stages, as well as the segregation of secretory and degradative compartments, relies on well-coordinated and directed movement within the cell immediately after cargo uptake from the extracellular space. Hereby, the endosomal system relies on bidirectional movement carried out by both kinesins and dyneins. Both of these often engage in a “tug of war” on the same cargo molecule. This tug of war helps position organelles correctly and determines the direction of cargo transport [14]. Bidirectional movement, especially in the early endosome, is considered crucial not only for endosome maturation but also for fate decision on endocytosed cargo whether it is destined for degradation or recycling [20]. One EE may harbor cargo that undergoes either degradation and recycling, necessitating a decision to be made at a specific time point. Consequently, the cargos destined for each fate undergo redistribution accordingly. The kinesin-1, kinesin-2 and kinesin-3 families are the primary players in endosomal transport processes. Strikingly, these three families employ distinct mechanochemical strategies to compete effectively with dynein during bidirectional transport. Thereby, it was shown that although members of the kinesin-2 and kinesin-3 family have a higher load sensitivity resulting in higher detachment rates from a specific cargo, they effectively outcompete dynein by showing faster reattachment rates [21].

Transport of early endosomal compartments

Transport of early endosomal compartments involves retrograde movement towards the perinuclear area, facilitated by dynein motors and minus-end directed kinesins. Phagocytosed endosomes subsequently fuse with existing sorting endosomes characterized by the presence of the small GTPase Rab5 [22]. Rab GTPases are vital regulators of membrane trafficking or organelle biogenesis. Upon GTP binding, they interact specifically with vesicles or membranes, recruiting specific effectors to facilitate downstream signaling or transport [23].

The early endosome has two potential outcomes: it can further mature into a late endosome positive for Rab7 or the cargo destined for recycling (i.e. cell surface receptors) are released from the EE with the help of Rab4 and Rab11 and subsequently enter the early recycling compartments [20, 24, 25]. Kif13A, a member of the kinesin-3 family, is frequently associated with recycling endosomes due to its interaction with Rab11. Kif13A plays a significant role in generating tubular structures representing recycling endosomes, engaging in a tug-of-war with dynein, and relying on the association with Rab22A [26, 27]. Overexpression of fluorescently tagged Kif13A even stabilizes the tubular structures representing recycling endosomes in HeLa cells [25, 27, 28]. Furthermore, it has been discovered that the small GTPase Rab10, together with Kif13A and Kif13B, is required for the generation of tubular recycling endosomes in HeLa cells [18]. Interestingly, both Kif13A and Kif13B also interact with Rab4-bearing vesicles as well and participate in the transport of vesicles towards the synapse together with a kinesin-2 motor in Drosophila [23, 29]. In C. elegans Kif13A, but not Kif13B is required for the transport of AMPA receptor-carrying vesicles marked with Rab11 towards the synapse during long-term potentiation (LTP) [30]. It is well established, that Kif13A and Kif13B preferentially interact with early endosomal compartments [31]. Recently, it was discovered that BLOS1, a subunit of BLOC-1 required for the generation of cell-type-specific lysosome-related organelles, tightly regulates the recycling of low-density lipoprotein receptors in hepatocytes by coordinating the switch between kinesin-2 motor Kif3B and kinesin-3 motor Kif13A based anterograde transport of REs [32]. Additionally, the recycling of internalized vascular endothelial growth factor receptor 2 (VEGFR2) through Rab11-positive recycling vesicles mediated by the activity of Kif13B plays a role in the regulation of endothelial permeability [33]. Another member of the kinesin-3 family, Kif16B, is also involved in the trafficking and tubulation of early endosomes [9].

Transport of late endosomal compartments

As endosomal compartments mature, their composition of surface proteins changes, which leads to alterations in their interactions with different motor proteins including kinesins. The transition from early to late endosomes is marked by the presence of Rab7a and Rab7b [20]. During this maturation process, cytoplasmic dynein becomes crucial for the transport of late endosomes towards the perinuclear area where they often fuse with lysosomes for degradation. Rab7 hereby interacts with multiple Rab-interacting proteins thus recruiting the dynactin-dynein complex required for retrograde transport to the late endosome or lysosome [34, 35]. While dynein is primarily responsible for the retrograde transport of late endosomes, recent research has also uncovered interactions between kinesin motors and late endosomal compartments. For example, retrograde transport of early endosomes has been shown to depend on the concerted antagonistic action of dynein and the kinesin motor KifC1 [36]. This indicates that kinesins can still play a role in the transport of endosomal compartments even at later stages of maturation.

Even lysosomes exhibit bidirectional movement within the cell, suggesting the involvement of both dynein and kinesin motors in their correct positioning. The activation of Rab7 leads to the recruitment of the effector FYCO1, which, together with phosphatidylinositol 3-phosphate (PI(3)P), recruits the kinesin-1 motor to the lysosome to ensure its correct positioning [34]. In HeLa cells, BORC and Arl8a/b have been identified as master regulators of lysosomal positioning. They recruit Kinesin-1 (Kif5B) as well as the kinesin-3 family members Kif1Bβ and Kif1A to move the lysosome along specific microtubule tracks [37]. Kif1A and Kif1B are known to prominently interact with late endosomal compartments [31]. In addition, Arl8b, another small GTPase, recruits Kinesin-1 to late endosomes/lysosomes for anterograde trafficking, while its interaction with the effector RUFY3 supports retrograde transport of lysosomes [38, 39]. This highlights the involvement of kinesins in bidirectional movement and positioning.

In summary, while dynein is primarily responsible for retrograde transport in endosomal maturation and lysosomal fusion, kinesin motors such as KifC1, Kif1A, Kif1B, Kif13A, Kif13B and Kif5B also participate in the transport and positioning of endosomal compartments and lysosomes at different stages of maturation. The interaction between specific Rab GTPases and their effectors helps recruit the appropriate motor proteins to ensure proper cargo sorting and organelle distribution within the cell (Fig. 1).

Concluding remarks and perspectives

The impact of kinesins on the positioning and movement of different endosomal compartments appears to be quite relevant. Not only do current results indicate that kinesins are implicated in the transport of early as well as late endosomal compartments but also constantly undergo a tug-of-war with cytoplasmic dynein. The constant competition of dynein and kinesin ensures not only the correct positioning of endosomes at any given time point but also maintains the balance of cargos destined for either recycling, degradation, or secretion and thus the homeostasis of the cell. While much is known about the participation of different kinesins in recycling and degradative pathways, the secretion of sEVs from late endosomal compartments also relies on the involvement of molecular motors, likely from the kinesin family, given the anterograde nature of this transport route.

Prior investigation in the fruit fly Drosophila melanogaster revealed the involvement of the kinesin-3 motor Klp98A in apical to basal transport of the exosomal cargo wingless (Wg) in the wing imaginal disc epithelium [40]. Previous findings indicate that Kif13A and Kif13B primarily interact with early endosomal compartments, while Kif1A and Kif1B primarily interact with late endosomal compartments [31]. Taken together, our own results support the idea that endosomes comprise of distinct subsets that are being transported by a number of different kinesins, as has already been reported by others [41]. Unraveling the network of kinesins and their potential adaptors acting together to facilitate anterograde transport and membrane fusion will be of great interest for discovering how secretory vs. degradative transport routes are chosen. Interestingly, potential adaptors also rely on the involvement of Rab GTPases such as Rab27a/b [42].

Given the substantial interest in sEVs as mediators in health and disease and the potential utilization of sEVs as potential biomarkers or nanocarriers, unraveling the intracellular network governing this type of intracellular communication is crucial. Therefore, it is important to identify the motor proteins involved, potential adaptors, and trafficking routes toward the plasma membrane that ultimately facilitate the release of sEVs.