A team of scientists at TIFR Mumbai takes us through their journey into the movements within cells.

As we breathe, eat, sleep, and live our daily lives, deep within the cells in our body, tiny molecular motors drive movements of intracellular organelles keeping the cells active. These molecular motors that generate energy for the movements by breaking down ATP are called molecular proteins.

Research in the Lab of Roop Mallik at the Tata Institute of Fundamental Research in Mumbai investigates how motor proteins drive such movements of organelles like endosomes, phagosomes and lipid droplets. The Mallik lab’s peek into the world of macroscopic motion helped decode the mechanism of movement of organelles, providing some progress in understanding neurodegenerative diseases and the effect of obesity on cells.

The interior of cells resemble a rather busy railway junction with goods trains shunting in and out. Proteins and lipids are packed into tiny packets called ‘Endosomes’ which move around rapidly and fuse with ‘lysosomes’, similarly, mitochondria moves to places where energy is required etc.

At first glance, this motion can appear chaotic, but there is order in this apparent chaos imposed by specific tracks, called the cytoskeleton of the cell, along which motion occurs. There are two major types of cytoskeleton – actin filaments and microtubules.

The ‘motor’ proteins move along the cytoskeletal tracks just like engines move along railway tracks driving wagons with cargo. The motor proteins generally contain 3 domains called head, neck and tail. The head domain binds the cytoskeleton and breaks down ATP to generate mechanical energy to move. The tail domain is required to attach specific cargo (e.g. endosomes, mitochondria etc) to the motor protein, and the neck domain helps in regulation of motor protein activity. Dynein and kinesin are two such motor proteins which move along microtubules.

It is known that microtubule ends are distinct (called plus and minus). The plus end of microtubules is usually towards the cell periphery whereas the minus end is close to the nucleus (near the microtubule organizing centre). Kinesin is a plus enddirected motor. This means it travels towards the microtubule plus ends. But dyne in is a minus end directedmotor. Together these two motors coordinate movement along microtubules and distribute many components to required locations inside cells.

Delving into the motion of endosomes, phagosomes and lipid droplets along microtubules, we described how the direction of motion of endosomes is controlled inside cells. We found that kinesin and dynein motors generate force to oppose each other, and at any given instant the direction of motion is determined by the winner of a ‘tug of war’ between these motors (Soppina et al, PNAS 2009).

We next moved our attention to ‘Phagosomes’ that are formed when cells engulf foreign particles. Macrophages (a class of white blood cells) engulf and destroy bacteria by enclosing them in phagosomes. Phagosome formation is also observed in unicellular amoeba. This enabledus to use the amoeba Dictyostelium discoidum to study motion of phagosomes (Rai et al Cell 2013; Rai et al Cell 2016). We showed that dynein motors can team up to generate large forces on phagosomes.This may allow dynein to perform diverse cellular functions, although each individual dynein generates a small force. Thus, in a group of dyneins, the leading dynein slowed down allowing trailing ones to catch up to it leading to higher cooperativity in force generation.

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Dr. Roop Mallik & his lab members.

In another study, we showed that phagosomal movement is divided into two phases (Rai et al, Cell 2016). Early phagosomes which have been just engulfed into the cell show back-and-forth movement along the microtubules, hinting that both dynein and kinesin are responsible for its motion.

However, Latephagosomes exhibitmotion predominantly toward the minus end of microtubules, suggesting that dynein is the major motor protein on latephagosomes. We showed that as phagosomes mature over time, they acquire patches of cholesterol in their membrane. Many dyneins cluster on a single patch and can then generate sufficient force to drive motion exclusively towards the minus end of microtubules.

Of particular interest is that the parasite Leishmania donovani, causative agent of kala-azar, can hinder the formation of these cholesterol patches. This disruption may help the parasite to evade our immune system and cause disease. Later, using mathematical simulations and statistical arguments, we showed that the back and forth motion of early phagosomes can be described by the tossing of a fair coin, but late phagosomes used a biased coin (remember Sholay?) to move in unidirectional manner towards the cell centre (Sanghavi et al, 2018).

Another line of study in our Lab concerns how the liver functions to keep us healthy. The liver controls many metabolic processes such as breakdown and synthesis of fat, proteins, lipids etc. In our body, most fat is stored in adipose tissues, but fat metabolism is predominantly controlled by the liver. Imagine the liver as a post-office which collects the bulk of letters, processes them, and sends them to their designated locations.

The liver receives fat from adipose tissue and processes it to form very low density lipoproteins (VLDL), which is secreted into the blood from where it can be picked up and metabolized by other organelles. VLDL particles make up the serum triglycerideand cholesterol levels in our blood. High levels of VLDL can cause chronic health complications. Inside the body, fats/lipids are stored in lipid droplets. It was believed that lipid droplets are static reservoirs of fat that supply energy when required, e.g. under conditions of calorie restriction. However, later studies showed that these lipid droplets also move around inside cells with the help of motor proteins.

We started our work on lipid droplets accidentally. As a motor biology lab, we were interested in purifying endosomes from rat liver to watch them move on microtubules stuck to a cover slip. In trying to do this, we instead observed micron-sized lipid droplets moving on the microtubule driven by kinesin motors (Barak et al, Nature Methods 2013). Trying to understand why these lipid droplets move at all, we made rats fast so that the liver would receive more fat from adipose tissue and form additional lipid droplets. We then isolated the lipid droplets and found that the motility of lipid droplets in fasted condition was reduced as compared to fed conditions. This suggested that the motion of lipid droplets inside liver depends on the metabolic state of the body.

We found a massive accumulation of lipid droplets inside liver cells in fasted condition as compared to fed condition, but the serum triglyceride level remained same in fed and fasted condition. This suggested an unknown mechanism inside the liver that maintains fat homeostasis during metabolic changes. In the absence of this mechanism, a massive surge of toxic fat would be released into blood every time we fast – thankfully, this does not happen. We tried to answer this question using the previous finding that lipid droplets are less motile in fasted condition compared to fed condition. Perhaps the reduced motion of lipid droplets was responsible for maintaining fat homeostasis? Microscopic studies showed that lipid droplets are localized at the periphery of cells inside the liver and this required kinesin driven motion of lipid droplets (Rai et al, PNAS 2017). But, why is this motion important? The answer to these questions came from studies which showed that the major enzymes required for VLDL assembly are present in the smooth endoplasmic reticulum that is localized towards the periphery in liver cells. So, kinesin is the delivery-boy that transports lipid droplets to the smooth endoplasmic reticulum, so that VLDL particles can be manufactured there using the fat inside these droplets.

This brought us to the next question, how is kinesin recruited to lipid droplets, and why does this change across metabolic states? Currently, we are trying to address these questions. Our hope is to develop a drug to control serum triglyceride level as a therapeutic strategy against hyperlipidaemia. Our work on phagosomes also has implications on human health. For example, dynein is known to be crucial in neurons and our findings may be able to shed light on neurodegenerative diseases. As another example, obesity, which causes higher amounts of cholesterol in the body, can adversely affect cellular processes by changing dynein activity.

Roni Saiba and Subham Kumar Tripathy, Roop Mallik’s group, TIFR-Mumbai
Dr Roop Mallik received the Infosys Prize 2018 for Life Sciences
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